Compositions produced using enteric pathogens and methods of use

ABSTRACT

The present invention provides compositions including polypeptides having the characteristics of polypeptides expressed by a reference microbe such  E. coli  or  Salmonella . Examples of  Salmonella  strains that can be used include, for instance,  S. enterica  serovar Newport,  S. enterica  serovar Enteritidis,  S. enterica  serovar Typhimurium, and  S. enterica  serovar Dublin. Also provided are compositions including polypeptides having a particular molecular weight and a mass fingerprint that includes polypeptide fragments having a particular set of masses, or polypeptides having an amino acid sequence with at least about 95% identity with an amino acid sequence, wherein the polypeptide has seroreactive activity. The present invention also provides methods of making and methods of using such compositions.

RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 15/279,595, filed on Sep. 29, 2016, which is acontinuation patent application of U.S. patent application Ser. No.14/798,726, filed on Jul. 14, 2015, now U.S. Pat. No. 9,463,230; whichis a divisional patent application of U.S. patent application Ser. No.12/269,636, filed on Nov. 12, 2008, now U.S. Pat. No. 9,109,028; whichis a divisional of U.S. patent application Ser. No. 10/946,647, filedSep. 20, 2004, now U.S. Pat. No. 8,119,147; which claims the benefit ofU.S. Provisional Application No. 60/504,119, filed Sep. 19, 2003, eachof which is incorporated by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext filed entitled “2015-07-13-SequenceListing.txt” having a size of622 kilobytes and created on Jul. 13, 2015. Due to the electronic filingof the Sequence Listing, the electronically submitted Sequence Listingserves as both the paper copy required by 37 CFR § 1.821(c) and the CRFrequired by § 1.821(e). The information contained in the SequenceListing is incorporated by reference herein.

BACKGROUND

The transmission of enteric pathogens to human populations by theconsumption of contaminated food and water has become a world wideconcern. Surveillance data compiled by the World Health Organizationestimate that gastrointestinal infections and their sequelae result inapproximately 4 million to 6 million deaths annually. More than 80% ofthese cases are among children under the age of five with mortalityreaching 4 million. The majority of these deaths are in children lessthan 2 years of age. In the United States, diarrhea is the second mostcommon infectious illness, accounting for one out of every sixinfectious diseases. In some developing countries, children have morethan 12 episodes of diarrhea per year and diarrheal diseases account for15 to 34 percent of all deaths.

In the United States food/waterborne diseases cause approximately 76million illnesses, 325,000 hospitalizations, and 5000 deaths each year.More than 90% of the foodborne illnesses of known causes are ofmicrobial origin. Costs associated with medical expenses and losses inproductivity associated with microbial agents are estimated to bebetween $5.6 and $9.4 billion dollars annually. The most commonlyrecognized food/borne pathogens contributing to gastrointestinalinfections have been shown to be bacteria (e.g., Salmonella spp.,Escherichia coli, Shigella spp., and Vibrio spp.).

The virulence and pathogenesis of enteric pathogens involves both hostand pathogen specific factors. Many pathogen-specific virulencedeterminants contribute to the pathogenesis of these bacteria. Thebacterial virulence of these bacteria is the result of many differentattributes, which often contribute to different steps in the complicatedseries of events we recognize as an infection. Infection occursprimarily by the consumption of contaminated water, food or by directperson to person contact. Once ingested the stages of infection commonto enteric pathogens can include attachment, colonization,proliferation, tissue damage, invasion and dissemination. Lessfrequently, enteric pathogens can produce a bacteremic conditioninducing reactive arthritis, kidney failure, Guillian-Barre, Reitersyndrome and other extra-intestinal symptoms.

The first host barrier that enteric pathogens must overcome is themucosal surface. A single epithelial cell layer separates the host fromthe lumen of the gastrointestinal tract. This barrier and a plethora ofother host antimicrobial mechanisms deter commensal, opportunistic andpathogenic microorganisms from establishing infection. Enteric pathogenshave evolved some elaborate pathogenic strategies to attach, invade andtranslocate across the gut epithelium to cause infection. Adherence tomucosal surfaces is a prerequisite of most enteric pathogens toestablish infection. In its simplest form adherence or attachmentrequires two factors: a receptor and an adhesin. A number of specializedstructures (adhesins) have been identified in enteric pathogens thatenhance intestinal colonization of the organism. These specializedstructures (e.g., pili or fimbriae) act as ligands to bind the bacterialcell to specific complex carbohydrate receptors on the epithelial cellsurface of the intestine. Once colonization is established entericpathogens have a multitude of virulence factors that enhance the abilityof the pathogen to invade its host. One of the more pronounced clinicalmanifestations of intestinal colonization is diarrhea. This clinicalsyndrome is typically induced by the synthesis and excretion of avariety of enterotoxins, (e.g., heat-labile toxin (LT), heat-stabletoxin (ST) cholera toxin (CT) and shiga toxin (Stx)) that cause a netsecretion of fluid and electrolytes (diarrhea). Many other specificvirulence factors of enteric pathogens have been described that affect awide range of eukaryotic cell processes in the host, to includinginvasion of specific cell types, cell to cell interactions and signaltransduction by integrins, attaching and effacing with destruction ofthe epithelial surface, elaboration of exotoxins, and actinpolymerization enhancing cell to cell spread, etc.

The diversity of enteric pathogens and virulence factors has complicatedthe development of new and improved vaccines with long lastingprotection. The search for a better vaccine is prompted by the resultsof epidemiological and challenge studies showing that the recovery fromnatural infection is often followed by long lasting immunity whileproviding cross-protection against multiple strains and/or serotypes.

Current vaccines under development for such enteric pathogens as Vibriocholera, Escherichia coli, Salmonella, and Shigella are based onparenteral and oral vaccines. Moderately effective vaccines have beentested and implemented for controlling cholera. The oral vaccinescurrently under development include two types: killed Vibrio cholerabacteria that are combined with purified cholera B subunit toxin, andlive-attenuated strains of V. cholera with known genetic deletions(Butterton et al., Infect. Immun. 65: 2127-2135). Field trials sponsoredby The World Health Organization using an oral vaccine consisting of awhole-cell B subunit reported levels of only 50% protection in humanpopulations in underdeveloped countries. The vaccine required multipledoses over a four month period; unfortunately, young children were notwell protected (Sack et al. Infect. Immun. 66:1968-1972 (1998); Sanchezet al, Lancet. 349: 1825-1830 (1997); and Trach et al. Lancet. 349:231-235 (1997)). A whole-cell vaccine containing four common isolates ofV. choleraa not containing B subunit toxin has also been tested in humansubjects that showed a protective efficacy of 65% (Taylor et al. Infect.Immun. 65: 3852-3856 (1997)). A whole-cell vaccine containing fourcommon isolates of cholera not containing B subunit toxin has also beentested in human subjects (Taylor et al. Infect. Immun. 65: 3852-3856(1997)). The vaccine required two administrations 7-14 days apart andinduced a protective index of approximately 65%. However, the vaccinewas not well tolerated due to its reactve nature upon injection. Severallive-attenuated vaccine candidates have been tested in large scaleefficacy trials involving more than 60,000 human subjects.Unfortunately, the results of this pivotal trial did not demonstrate theeffectiveness of the vaccine in preventing cholera. Further developmentin live attenuated gene deleted vaccines has recently shown promiseagainst the 01 and 0139 serotypes in human volunteers. However, efficacyof the vaccine in large populations and protection against multipleserotypes have yet to be demonstrated.

There are five categories of diarrheagenic Escherichia coli that causefoodborne and waterborne diseases in humans: the enteropathogenic(EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroinvasive(EIEC) and enteroaggregative (EAEC) strains. The mechanism of diseaseassociated with these pathogens depends on specific characteristicswhich involve attaching and effacing adherence of the organism tointestinal epithelial cells and damage to the intestinal microvilli. Ofparticular interest has been the emergence of the Shiga toxin-producingE. coli, also referred to as EHEC, primarily of the O157:H7 serotype.This strain of E. coli has been shown to synthesize either one or bothof the Shiga toxins (Stx-1 and/or Stx-2). This strain has beenassociated with gastrointestinal infections that begin with diarrheathat can exasperate into hemorrhagic colitis, followed byhemolytic-uremic syndrome (HUS) and/or encephalopathy, particularly inthe young, immunocompromized, and elderly adults. The Shiga toxin (Stx)produced by this isolate is believed to be important in the pathogenesisof this organism. Current efforts at vaccine development are primarilyfocused on animals known to asymptomatically carry these organisms andshed them in their feces. Research has focused on a number of strategiesfor controlling this organism, which revolve around the concept ofpreventing colonization by targeting the colonization factor intimin,and immunization of animals with genetically modified non-toxinproducing versions of the parent isolate. The intimin protein has beenshown to be responsible for the attaching and effacing lesions alsocharacteristic of both Shigella dysenteriae (STEC) and theenteropathogenic (EPEC) strains of E. coli. In addition, researchershave been investigating the expression of intimin in animal feedproducts such as canola and alfalfa for use as an edible animal vaccine.If any of these strategies work in animals it could find its way tohuman usage (Acheson et al. Infect. Immun. 64: 355-357 (1996); Bokete etal. J. Infect. Dis. 175: 1382-1389 (1997); Bosworth et al. Infect.Immun. 64:55-60 (1996) and Konadu et al. Infect. Immun. 62: 5048-5054(1964)).

The National Institute of Child Health and Human development haveproposed the use of conjugate vaccines using the B-subunit of Stx-1 inconjunction with a whole cell as developed for V. cholerae, which hasshown promising results in experimental animal models as well as toxoidsand immunotherapeutics using antitoxin antibodies as well as humanmonoclonal antibodies to neutralize the Stx-1 and Stx-2 toxin. Suchprophylactic and immunotherapeutic strategies could protect against STECinfection as well as infections caused by closely related organisms suchas EPEC and EHEC strains of E. coli.

Enterotoxigenic (ETEC) strains of E. coli are an important cause ofdiarrhea in infants in less developed countries. It is estimated thatETEC causes more than 650 million cases of diarrhea per year and morethan 800,000 deaths in children less than 5 years of age. ETEC is alsothe major cause of traveler's diarrhea, which affects at least 8 millionUnited States citizens who travel to endemic regions of the world eachyear. Virulence factors associated with these strains of E. coli includeprimarily adhesins and enterotoxins such as LT1, STa and STb. Involunteer studies infection with ETEC generates protective immunityagainst rechallenge with the same strain. The vaccine candidatecurrently being developed consists of a mixture of fiveformalin-inactivated ETEC strains, which together express the requiredadhesins, combined with a recombinant Cholera toxin B subunit, whichgenerates antibody that cross-reacts with the ETEC-LT toxin. Clinicalstudies have shown that the vaccine is immunogenic and safe in humanvolunteers.

Shigella spp. such as S. sonnei, S. flexneri, S. boydii and S.dysenteriae are causative agents of shigellosis or bacillary dysentery.In the United States approximately 13,000 cases of shigellosis werereported in 2002, a 22% increase from 2001 (CDC, Shigella Annual Summary2002). Nearly 30% of the reported cases occurred in children under theage of five. The mechanism of disease associated with these pathogens ischaracterized by specific attaching and effacing lesions involvingmicrovilli destruction, and the production of potent exotoxins (Shigatoxin) that frequently results in hemolytic uremic syndrome. A virulenceplasmid present in all invasive Shigella strains has been identifiedthat encode a number of outer membrane proteins that mediate attachmentto the epithelial cell. Several of the plasmid-encoded proteins initiateparasite-induced phagocytosis which in turn breaks down the membrane ofthe phagocytic vacuole, allowing bacteria to multiply within thecytoplasm.

Vaccine strategies created to control shigellosis have focused onattenuated strains with known genetic deletions. A deletion mutant of S.flexneri has shown excellent protection after a single oral dose. Thisvaccine candidate provides protection against severe shigellosis involunteers challenged with S. flexneri. Other vaccine strategies includethe development of auxotrophic mutants and recent studies have shownprotection using O-specific polysaccharides conjugates from S. sonneiand S. flexneri. As with many of these diseases a comprehensive vaccineapproach to controlling shigellosis must include various bacterialcomponents to protect against the multiple serotypes of Shigella thatare responsible for endemic outbreaks of dysentery (Ashkenazi et al., J.Infect. Immun. 179: 1565-1568 (1999); Cohen et al., Lancet. 349: 155-159(1997); Coster et al., Infect. Immun. 67: 3437-3437 (1999); Kotloff etal., infect Immun. 64: 4542-4548 (1996) and Sansonetti et al., Res.Immunol. 147:595-602 (1996)).

Salmonella infections are the leading cause of bacterial foodbornediseases worldwide and are one of the most common enteric diseases inthe United States. There are approximately 2,213 different Salmonellastrains currently identified which can be classified according to theiradaptation to human and animal hosts. For instance, S. typhi and S.paratyphi causes enteric or typhoid fever only in humans and globallyinfect 20-30 million people annually and cause 600,000 deaths. In theUnited States, more than 41,000 cases were reported in 1993 with thehighest incidence being in children 5 to 19 years of age. Non-typhoidalSalmonella enterica is one of the most common causes of food poisoningin the United States, responsible for an estimated 1.4 million cases ofsalmonellosis annually (Mead et al. Emerg. Infect. Dis. 5:607-625(1999)). The cost of human salmonellosis in the U.S. is estimated to beseveral billion dollars annually based on healthcare costs and lostproductivity.

There has been a number of virulence factors associated with diseasecaused by Salmonella. Briefly, the pathogenesis of the organism beginswith the colonization of the host followed by localized degeneration ofthe epithelial surface resulting in penetration of the epithelialbarrier and proliferation in the lamina propria, multiplication, andstimulation of an inflammatory response. Diarrhea associated withsalmonellosis is associated primarily with the inflammatory response,which stimulates the release of prostaglandins and production of cAMP,which increase the secretion of fluid and electrolytes into the lumen ofthe bowel (diarrhea).

A number of parenteral whole-cell vaccines for typhoid fever have beendeveloped but have been found to be only marginally effective because ofsevere adverse reactions in vaccinates. Currently the National Instituteof Child Health and Human Development has developed and tested a vaccineconsisting of the Vi antigen. Clinical trials have demonstrated anefficacy of 72-80% with a single injection. A number of gene deletedmutants have been developed for controlling S. typhi with varyingdegrees of success (Germanier et al. J. Infect. Dis 131:553-558 (1975);Hohmann et al. J. Infect. Dis. 173:1408-1414 (1996); Nardelli-Haefligeret al. Infect. Immun. 64:5219-5224 (1996); Stocker et al. Vaccine.6:141-145 (1988); Szu et al. Infect. Immun. 62: 4440-4444 (1964); Tacketet al. Infect Immun. 60: 536-541 (1992); and Tacket et al. Vaccine. 10:443-446 (1992)).

The remaining Salmonella strains commonly referred to as nontyphoidalare primarily transmitted from animals to humans (Calnek et al.,Diseases of Poultry-9^(th) ed., pp. 99-130, Iowa State University, AmesIowa (1991)). In the United States, the most common serotypes of S.enterica isolated from humans are serotypes Typhimurium, Enteritidis,and Newport (CDC Salmonella Annual Summary, 2002). These three serotypesaccounted for 51% of human Salmonella isolates in 2002. Notably, theserotypes Typhimurium and Newport are frequently resistant to multipleantibiotics. In a 2001 annual survey, 53% of Typhimurium isolates wereresistant to at least one antibiotic and 30% were resistant to fiveantibiotics in a manner characterisitic of the DT104 phage type (CDCNational Antimicrobial Resistance Monitoring System:Enteric Bacteria,available at www.cdc.gov/narms/). In addition, 26% of Newport isolateswere resistant to at least nine antibiotics in the 2001 annual survey.The Typhimurium and Newport serotypes are primarily associated with theconsumption of a variety of different types of animal products thatbecome contaminated during processing or handling. In contrast,Salmonella serotype Enteritidis is almost exclusively associated withthe consumption of contaminated chicken eggs. This serotype has apropensity to colonize poultry ovarian tissues for extended periods oftime (Okamura et al., Avian Dis., 45: 61-69 (2001) and Okamura et al.,Avian Dis., 45: 962-971 (2001)), and can gain entry to the eggenvironment by vertical transmission during egg formation (Gast et al.,Avian Dis. 44: 706-710 (2000) and Humphrey et al., Int. J. FoodMicrobiol. 21: 31-40 (1994)). A recent risk assessment estimated that2.3 million eggs are contaminated in the United States annually,resulting in approximately 660,000 human infections (Hope et al., RiskAnal., 22:203-218 (2002)). Additional serotypes that have beenassociated with human salmonellosis derived from poultry and otheranimals include S. enterica Heidelberg, Hadar, Infantis, Agona,Montevideo, Thompson, and Braenderup.

Research for controlling nontyphoidal Salmonella has been primarilylimited to the bacterins, which consist of killed Salmonella cells, andthe live attenuated strains of Salmonella. Bacterins typically stimulateantibody responses in vaccinated animals but may be limited in theirability to promote cell-mediated immunity (Babu et al., Vet. Immunol.Immunopathol. 91:39-44 (2003) and Okamura et al., Comp. Immunol.Microbiol. Infect. Dis. 27:255-272 (2004)), an important host responsefor effective clearance of Salmonella (Lalmanach and Lantier. MicrobesInfect. 1:719-726 (1999) and Naiki et al., J. Immunol. 163:2057-2063(1999)). In addition, bacterins have generally produced inconsistentprotection against fecal shedding of Salmonella (House et al., Am. J.Vet. Res. 12: 1897-1902 (2001) and Davison et al., Avian Dis. 43:664-669(1999)). Other disadvantages of bacterins include injection-sitegranulomas, weight loss, and serotype-specific protection. The liveattenuated Salmonella vaccines are generally considered to providebetter cross-protection than observed with the bacterins (Hassan andCurtiss, III. Infect. Immun. 62:5519-5527. (1994)), and additionallystimulate both humoral and cell-mediated immune responses (Curtiss, IIIet al., Vet Microbiol. 37:397-405 (1993) and Villarreal-Ramos et al.,Vaccine 16: 45-54 (1998)). However, there are significant obstaclesregarding the safety of introducing these organisms into commercialanimals; specifically, there is concern that genetic reversion willoccur and render the vaccine strain virulent. A second potential problemwith using modified live vaccines is that antibodies generated to thesomatic antigen of the vaccination strains can interfere with nationaland state Salmonella monitoring programs by generating false positivereactions. In addition, antibiotics are often administered in commercialflocks to control infection rates which can eliminate the attenuatedvaccine strain; hence, repeated immunizations of live Salmonellavaccines are often required. There have been relatively few attempts toformulate subcellular vaccines for controlling Salmonella inagricultural animals. A few key studies in poultry species utilizedcrude cell extracts in their vaccinations, showing S.Enteritidis-specific mucosal and/or circulating antibody responses(Fukutome et al., Dev. Comp. Immunol. 25:475-484 (2001) andOchoa-Reparaz et al., Vet. Res. 35:291-298 (2004)). In other studies,purified outer membrane protein compositions were demonstrated topromote heightened antibody responses and reduced intestinalcolonization or fecal shedding following challenge with S. Enteritidis(Charles et al., Am. J. Vet. Res. 55:636-642 (1994), Khan et al., J.Appl. Microbiol. 95:142-145 (2003), and Meenakshi et al., Vet. Res.Commun. 23:81-90 (1999)).

SUMMARY

The present invention provides compositions including a polypeptidehaving the characteristics of a polypeptide expressed by a referencemicrobe. The characteristics of the polypeptide include both molecularweight and mass fingerprint. The reference microbe may be, for instance,an E. coli or a Salmonella. Examples of Salmonella strains that can beused include, for instance, S. enterica serovar Newport, S. entericaserovar Enteritidis, S. enterica serovar Typhimurium, and S. entericaserovar Dublin. Preferably, the reference polypeptide is expressed bythe microbe during growth in low metal conditions. The present inventionalso provides compositions including a polypeptide having a particularmolecular weight and a mass fingerprint that includes polypeptidefragments having a particular set of masses. The present inventionfurther provides compositions including a polypeptide having an aminoacid sequence with at least about 95% identity with a reference aminoacid sequence, wherein the polypeptide has seroreactive activity.

The compositions of the present invention may optionally include apharmaceutically acceptable carrier. The present invention also includesmethods for using the polypeptides disclosed herein. Methods includeinducing the production of antibody in an animal, treating a gramnegative microbial infection in an animal, and decreasing intestinalcolonization of an animal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The percent mortality in mice showing a dose response to varyingconcentrations of a composition prepared from Salmonella entericaserovar Newport after challenge. Non-diluted, 1/10, 1/100, and 1/1000refer to the dilution of the stock vaccine as described in Example 5.Numbers above the bars indicate the percent mortality.

FIG. 2. The difference in fecal shedding between vaccinated andnon-vaccinated mice after oral challenge with Salmonella entericaserovar Newport. Log₁₀ CFU, mean number of bacteria in fecal sample.

FIG. 3. The difference in fecal shedding between vaccinated andnon-vaccinated mice after oral challenge with E. coli O157:H7. Log₁₀CFU, mean number of bacteria in fecal sample.

FIG. 4. The serological response in vaccination with a compositionderived from S. enterica serovar Newport compared to non-vaccinatedcontrols as evaluated by ELISA.

FIG. 5. The difference in fecal shedding between vaccinated andnon-vaccinated calves after oral challenge with a composition derivedfrom S. enterica serovar Newport.

FIG. 6. The difference in fecal shedding between vaccinated andnon-vaccinated steers after oral challenge with a nalidixic acidresistant E. coli O157:H7.

FIG. 7. The difference in fecal shedding between vaccinated andnon-vaccinated steers after oral challenge with a nalidixic acidresistant E. coli O157:H7.

FIG. 8. Clearance of Salmonella enterica serovar Enteritidis from theSpleens of Control and Vaccinated Groups following Intravenous Challengein Chickens.

FIG. 9. Clearance of Salmonella enterica serovar Enteritidis from theOvaries of Control and Vaccinated Groups following Intravenous Challengein Chickens.

FIG. 10. Difference in Fecal Shedding of Salmonella enterica serovarEnteritidis in Control and Vaccinated Groups following IntravenousChallenge in Chickens.

FIG. 11. Comparison of selected proteins identified using E. coli grownunder iron-limiting conditions with proteins from other pathogens.

FIG. 12. Nucleotide sequence of SEQ ID NO:1367.

FIG. 13. Nucleotide sequence of SEQ ID NO:1368.

FIG. 14. Nucleotide sequence of SEQ ID NO:1369.

FIG. 15. Nucleotide sequence of SEQ ID NO:1370.

FIG. 16. Nucleotide sequence of SEQ ID NO:1371.

FIG. 17. Nucleotide sequence of SEQ ID NO:1372.

FIG. 18. Nucleotide sequence of SEQ ID NO:1373.

FIG. 19. Nucleotide sequence of SEQ ID NO:1374.

FIG. 20. Nucleotide sequence of SEQ ID NO:1375.

FIG. 21. Nucleotide sequence of SEQ ID NO:1376.

FIG. 22. Nucleotide sequence of SEQ ID NO:1377.

FIG. 23. Nucleotide sequence of SEQ ID NO:1378.

FIG. 24. Nucleotide sequence of SEQ ID NO:1379.

FIG. 25. Nucleotide sequence of SEQ ID NO:1380.

FIG. 26. Nucleotide sequence of SEQ ID NO:1382.

FIG. 27. Nucleotide sequence of SEQ ID NO:1383.

FIG. 28. Nucleotide sequence of SEQ ID NO:1384.

FIG. 29. Nucleotide sequence of SEQ ID NO:1385.

FIG. 30. Nucleotide sequence of SEQ ID NO:1386.

FIG. 31. Nucleotide sequence of SEQ ID NO:1387.

FIG. 32. Nucleotide sequence of SEQ ID NO:1388.

FIG. 33. Nucleotide sequence of SEQ ID NO:1389.

FIG. 34. Nucleotide sequence of SEQ ID NO:1390.

FIG. 35. Nucleotide sequence of SEQ ID NO:1391.

FIG. 36. Nucleotide sequence of SEQ ID NO:1392.

FIG. 37. Nucleotide sequence of SEQ ID NO:1393.

FIG. 38. Nucleotide sequence of SEQ ID NO:1394.

FIG. 39. Nucleotide sequence of SEQ ID NO:1395.

FIG. 40. Nucleotide sequence of SEQ ID NO:1396.

FIG. 41. Nucleotide sequence of SEQ ID NO:1397.

FIG. 42. Nucleotide sequence of SEQ ID NO:1398.

FIG. 43. Nucleotide sequence of SEQ ID NO:1399.

FIG. 44. Nucleotide sequence of SEQ ID NO:1400.

FIG. 45. Nucleotide sequence of SEQ ID NO:1401.

FIG. 46. Nucleotide sequence of SEQ ID NO:1402.

FIG. 47. Nucleotide sequence of SEQ ID NO:1403.

FIG. 48. Nucleotide sequence of SEQ ID NO:1404.

FIG. 49. Nucleotide sequence of SEQ ID NO:1405.

FIG. 50. Nucleotide sequence of SEQ ID NO:1406.

FIG. 51. Nucleotide sequence of SEQ ID NO:1407.

FIG. 52. Nucleotide sequence of SEQ ID NO:1408.

FIG. 53. Nucleotide sequence of SEQ ID NO:1409.

FIG. 54. Nucleotide sequence of SEQ ID NO:1410.

FIG. 55. Nucleotide sequence of SEQ ID NO:1411.

FIG. 56. Nucleotide sequence of SEQ ID NO:1412.

FIG. 57. Nucleotide sequence of SEQ ID NO:1413.

FIG. 58. Nucleotide sequence of SEQ ID NO:1414.

FIG. 59. Nucleotide sequence of SEQ ID NO:1415.

FIG. 60. Nucleotide sequence of SEQ ID NO:1416.

FIG. 61. Nucleotide sequence of SEQ ID NO:1417.

FIG. 62. Nucleotide sequence of SEQ ID NO:1418.

FIG. 63. Nucleotide sequence of SEQ ID NO:1419.

FIG. 64. Nucleotide sequence of SEQ ID NO:1420.

FIG. 65. Nucleotide sequence of SEQ ID NO:1421.

FIG. 66. Nucleotide sequence of SEQ ID NO:1422.

FIG. 67. Nucleotide sequence of SEQ ID NO:1423.

FIG. 68. Nucleotide sequence of SEQ ID NO:1424.

FIG. 69. Nucleotide sequence of SEQ ID NO:1425.

FIG. 70. Nucleotide sequence of SEQ ID NO:1426.

FIG. 71. Nucleotide sequence of SEQ ID NO:1427.

FIG. 72. Nucleotide sequence of SEQ ID NO:1428.

FIG. 73. Nucleotide sequence of SEQ ID NO:1429.

FIG. 74. Nucleotide sequence of SEQ ID NO:1430.

FIG. 75. Nucleotide sequence of SEQ ID NO:1431.

FIG. 76. Nucleotide sequence of SEQ ID NO:1432.

FIG. 77. Nucleotide sequence of SEQ ID NO:1433.

FIG. 78. Nucleotide sequence of SEQ ID NO:1434.

FIG. 79. Nucleotide sequence of SEQ ID NO:1435.

FIG. 80. Nucleotide sequence of SEQ ID NO:1436.

FIG. 81. Nucleotide sequence of SEQ ID NO:1437.

FIG. 82. Nucleotide sequence of SEQ ID NO:1438.

FIG. 83. Nucleotide sequence of SEQ ID NO:1439.

FIG. 84. Nucleotide sequence of SEQ ID NO:1440.

FIG. 85. Nucleotide sequence of SEQ ID NO:1441.

FIG. 86. Nucleotide sequence of SEQ ID NO:1442.

FIG. 87. Nucleotide sequence of SEQ ID NO:1443.

FIG. 88. Nucleotide sequence of SEQ ID NO:1444.

FIG. 89. Nucleotide sequence of SEQ ID NO:1445.

FIG. 90. Nucleotide sequence of SEQ ID NO:1446.

FIG. 91. Nucleotide sequence of SEQ ID NO:1447.

FIG. 92. Nucleotide sequence of SEQ ID NO:1448.

FIG. 93. Nucleotide sequence of SEQ ID NO:1381.

FIG. 94. The difference in mortality between vaccinated andnon-vaccinated chickens after intravenous challenge with S. entericaserovar Enteritidis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations, and the like. The term polypeptide does not connote aspecific length of a polymer of amino acids. A polypeptide may beobtainable directly from a natural source, or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. In the case of apolypeptide that is naturally occurring, such polypeptide is typicallyisolated. An “isolated” polypeptide is one that has been removed fromits natural environment. For instance, an isolated polypeptide is apolypeptide that has been removed from the cytoplasm or from the outermembrane of a cell, and many of the polypeptides, nucleic acids, andother cellular material of its natural environment are no longerpresent. A “purified” polypeptide is one that is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. Polypeptides that are producedoutside the organism in which they naturally occur, e.g., throughchemical or recombinant means, are considered to be isolated andpurified by definition, since they were never present in a naturalenvironment. As used herein, a “polypeptide fragment” refers to aportion of a polypeptide that results from digestion of a polypeptidewith a protease. Unless otherwise specified, “a,” “an,” “the,” and “atleast one” are used interchangeably and mean one or more than one.

A polypeptide of the present invention may be characterized by molecularweight. The molecular weight of a polypeptide, typically expressed inkilodaltons (kDa), can be determined using routine methods including,for instance, gel filtration, gel electrophoresis including sodiumdodecyl sulfate (SDS) polyacrylamide gel electrophoresis, capillaryelectrophoresis, mass spectrometry, and liquid chromatography includingHPLC.

A polypeptide of the present invention may be characterized by massfingerprint. As used herein, a “mass fingerprint” refers to a populationof polypeptide fragments obtained from a polypeptide after digestionwith a protease. Typically, the polypeptide fragments resulting from adigestion are analyzed using a mass spectrometric method. Eachpolypeptide fragment is characterized by a mass, or by a mass (m) tocharge (z) ratio, which is referred to as an “m/z ratio” or an “m/zvalue”. Methods for generating a mass fingerprint of a polypeptide areroutine. An example of such a method is disclosed in Example 25.

The polypeptides of the present invention may be metal regulatedpolypeptides. As used herein, a “metal regulated polypeptide” is apolypeptide that is expressed by a microbe at a greater level when themicrobe is grown in low metal conditions compared to growth of the samemicrobe in high metal conditions. Low metal and high metal conditionsare described herein. For instance, a metal regulated polypeptide is notexpressed at detectable levels during growth of the microbe in highmetal conditions but is expressed at detectable levels during growth inlow metal conditions. Another type of metal regulated polypeptide isexpressed at detectable levels during growth of the microbe in highmetal conditions but expressed at higher levels during growth in lowmetal conditions. The expression of such polypeptides is referred toherein as “enhanced” during growth in low metal conditions. In general,metal regulated polypeptides typically have a molecular weight of 66 kDaor greater. Polypeptides that are not metal regulated are typicallyexpressed at about the same level when the microbe is grown in low metaland high metal conditions. In general, non-metal regulated polypeptidestypically have a molecular weight of less than 66 kDa.

Whether a metal regulated polypeptide is expressed at a detectable levelor has enhanced expression during growth in low metal conditions can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a microbe are grown under highmetal conditions and under low metal conditions, polypeptides of thepresent invention are isolated as described herein, and the polypeptidespresent in each culture are resolved and compared. Typically, an equalamount of polypeptide from each culture is used. For instance, when SDSpolyacrylamide gel electrophoresis is used to compare the polypeptides,about 30 μg micrograms of polypeptide from each culture is used andloaded into a well. After running the gel and staining the polypeptides,the two lanes can be compared.

Preferably, polypeptides of the present invention have immunogenicactivity. “Immunogenic activity” refers to the ability of a polypeptideto elicit an immunological response in an animal. An immunologicalresponse to a polypeptide is the development in an animal of a cellularand/or antibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced.

In one aspect, a polypeptide of the present invention has thecharacteristics of a polypeptide expressed by a reference microbe. Thecharacteristics include both molecular weight and mass fingerprint. Thereference microbe can be Salmonella or an E. coli. Preferred examples ofeach of these are detailed in Table 1.

TABLE 1 Bacterial strains. Bacterial cell Laboratory designation S.enterica serovar Newport MS020508 S. enterica serovar EnteritidisMS010531 S. enterica serovar Typhimurium MS010427 S. enterica serovarDublin IRP SDC Serial E. coli BEcO157(stx-), MS040330, MS040324, orMS040827

When the reference microbe is S. enterica serovar Newport, for instanceMS020508, a candidate polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of 82 kDa, 80 kDa, 74kDa, 65 kDa, 56 kDa, 55 kDa, 52 kDa, 45 kDa, 38 kDa, 36 kDa, 22 kDa, 18kDa, or 12 kDa, and has a mass fingerprint that is similar to the massfingerprint of a polypeptide expressed by a reference microbe and havinga molecular weight of 80 kDa, 74 kDa, 65 kDa, 56 kDa, 55 kDa, 52 kDa, 45kDa, 38 kDa, 36 kDa, 22 kDa, 18 kDa, or 12 kDa, respectively.

When the reference microbe is S. enterica serovar Enteritidis, forinstance MS010531, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of 92 kDa, 91 kDa, 86kDa, 83 kDa, 78 kDa, 55 kDa, 40 kDa, 39 kDa, or 38 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight of 92kDa, 91 kDa, 86 kDa, 83 kDa, 78 kDa, 55 kDa, 40 kDa, 39 kDa, or 38 kDa,respectively.

When the reference microbe is S. enterica serovar Typhimurium, forinstance MS010427, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of 86 kDa, 82 kDa, 77kDa, 40 kDa, 39 kDa, or 38 kDa, and has a mass fingerprint that issimilar to the mass fingerprint of a polypeptide expressed by areference microbe and having a molecular weight of 86 kDa, 82 kDa, 77kDa, 40 kDa, 39 kDa, or 38 kDa, respectively.

When the reference microbe is S. enterica serovar Dublin, for instanceIRP SDC Serial, a polypeptide is considered to be a polypeptide of thepresent invention if it has a molecular weight of 96 kDa kDa, 89 kDa, 81kDa, 61 kDa, 56 kDa, 51 kDa, 43 kDa, 40 kDa, or 38 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight of 96 kDakDa, 89 kDa, 81 kDa, 61 kDa, 56 kDa, 51 kDa, 43 kDa, 40 kDa, or 38 kDa,respectively.

When the reference microbe is an E. coli, for instance BEcO157(stx-), apolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 90 kDa, 86 kDa, 83 kDa, 79 kDa, 66 kDa,56 kDa, 38 kDa, 37 kDa, or 29 kDa, and has a mass fingerprint that issimilar to the mass fingerprint of a polypeptide expressed by areference microbe and having a molecular weight of 90 kDa, 86 kDa, 83kDa, 79 kDa, 66 kDa, 56 kDa, 38 kDa, 37 kDa, or 29 kDa, respectively.

When the reference microbe is an E. coli, for instance MS040330, apolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 92 kDa, 80 kDa, 77 kDa, 72 kDa, 66 kDa,50 kDa, 42 kDa, 38 kDa, 36 kDa, 35 kDa, 30 kDa, 19 kDa, or 16 kDa, andhas a mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and having a molecularweight of 92 kDa, 80 kDa, 77 kDa, 72 kDa, 66 kDa, 50 kDa, 42 kDa, 38kDa, 36 kDa, 35 kDa, 30 kDa, 19 kDa, or 16 kDa, respectively.

When the reference microbe is an E. coli, for instance MS040324, apolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 88 kDa, 82 kDa, 79 kDa, 60 kDa, 54 kDa,46 kDa, 45 kDa, 38 kDa, 37 kDa, 31 kDa, 30 kDa, 19 kDa, 16 kDa, and hasa mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and having a molecularweight of 88 kDa, 82 kDa, 79 kDa, 60 kDa, 54 kDa, 46 kDa, 45 kDa, 38kDa, 37 kDa, 31 kDa, 30 kDa, 19 kDa, 16 kDa, respectively.

When the reference microbe is an E. coli, for instance MS040827, apolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 101 kDa, 88 kDa, 85 kDa, 77 kDa, 67 kDa,38 kDa, 35 kDa, and has a mass fingerprint that is similar to the massfingerprint of a polypeptide expressed by a reference microbe and havinga molecular weight of 101 kDa, 88 kDa, 85 kDa, 77 kDa, 67 kDa, 38 kDa,35 kDa, respectively.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide can beobtainable from a microbe, preferably a gram negative microbe, morepreferably, a member of the family Enterobacteriaceae, for instance, amember of the tribe Escherichieae or Salmonelleae. A candidatepolypeptide may also be produced using recombinant, enzymatic, orchemical techniques.

A candidate polypeptide may be evaluated by mass spectrometric analysisto determine whether the candidate polypeptide has a mass fingerprintsimilar to one of the polypeptides expressed by a reference microbe andreferred to above by molecular weight. Typically, the candidatepolypeptide is purified, for instance by resolving the candidatepolypeptide by gel electrophoresis and excising the portion of the gelcontaining the candidate polypeptide. Any gel electrophoresis methodthat separates polypeptides based on differing characteristics can beused, including 1 dimensional or 2 dimensional gel electrophoresis, aswell as separation based on, for instance, hydrophobicity, pI, or size.The candidate polypeptide is fragmented, for instance by digestion witha protease. Preferably, the protease cleaves the peptide bond on thecarboxy-terminal side of the amino acid lysine and the amino acidarginine, except when the amino acid following the lysine or thearginine is a proline. An example of such a protease is trypsin. Methodsfor digesting a polypeptide with trypsin are routine and known to theart. An example of such a method is disclosed in Example 24.

Methods for the mass spectrometric analysis of polypeptides are routineand known to the art and include, but are not limited to, matrixassisted laser desorption/ionization time of flight mass spectroscopy(MALDI-TOF MS). Typically, a mixture containing the polypeptidefragments obtained from a candidate polypeptide is mixed with a matrixthat functions to transform the laser energy to the sample and produceionized, preferably monoisotopic, polypeptide fragments. Examples ofmatrices that can be used include, for instance, sinapinic acid andcyano-4-hydroxycinnamic acid. An example of a method for the analysis ofpolypeptides by MALDI-TOF MS is described in Example 24. The ionizedpolypeptide fragments are separated according to their m/z ratio, anddetected to yield a spectrum of m/z ratio versus intensity. The spectrumincludes m/z values that represent the polypeptide fragments derivedfrom the candidate polypeptide. For any given polypeptide, the amount ofeach polypeptide fragment resulting from a trypsin digestion should beequimolar. However, it is known that trypsin digestion is not 100%efficient, for instance, some sites are more efficiently cleaved. Thus,when MALDI-TOF MS is used to determine m/z values, the intensity of eachm/z value is typically not identical. Generally, a spectrum has abackground level of noise present across most of the x-axis (i.e., theaxis having the values of the m/z ratios). This background level ofnoise varies depending on the running conditions and the machine used,and is easily identified by visual inspection of the spectrum. An m/zvalue is generally considered to represent a polypeptide fragment whenthe intensity is at least 2 times greater, 3 times greater, or 4 timesgreater than the background level of noise. The spectrum usuallyincludes other m/z values that are artifacts resulting from, forinstance, incomplete digestion, over digestion, other polypeptides thatmay be present in the mixture, or the protease used to digest thepolypeptide including m/z values resulting from autolysis of theprotease. This method of digesting a polypeptide with a protease isrecognized by the art as resulting in a mass fingerprint of greatspecificity that can be used to accurately characterize the polypeptideand distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide isanalyzed by mass spectroscopy, preferably both the candidate polypeptideand the polypeptide from the reference microbe are prepared and analyzedtogether, thereby decreasing any potential artifacts resulting fromdifferences in sample handling and running conditions. Preferably, allreagents used to prepare and analyze the two polypeptides are the same.For instance, the polypeptide from the reference microbe and thecandidate polypeptide are isolated under substantially the sameconditions, fragmented under substantially the same conditions, andanalyzed by MALDI-TOF MS on the same machine under substantially thesame conditions. A mass fingerprint of a candidate polypeptide isconsidered to be similar to the mass fingerprint of a polypeptide from areference microbe when 80%, 90%, 95%, or substantially all of the m/zvalues present in the spectrum of the reference microbe polypeptide andabove the background level of noise are also present in the spectrum ofthe candidate polypeptide.

In another aspect, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of a referencepolypeptide described in Table 2, 3, 4, 5, 6, 7, 8, or 9 and has a massfingerprint that includes the population of polypeptide fragments of thereference polypeptide as listed in Table 2, 3, 4, 5, 6, 7, 8, or 9. Forinstance, a polypeptide of the present invention includes a polypeptideof 82 kDa and a mass fingerprint that includes polypeptide fragmentshaving masses of 629.39, 644.37, 772.42, 831.45, 873.46, 991.55,1083.61, 1208.58, 1325.75, 1378.66, 1500.71, 1619.77, 1634.84, 1619.77,1728.83, 1872.88, 1981.96, 1998.06, 2193.94, and 2332.05. The massfingerprint of a candidate polypeptide can be determined by a massspectrometric method as described herein, preferably, by MALDI-TOF MS.The mass fingerprint of a candidate polypeptide will generally haveadditional polypeptide fragments and therefore additional m/z valuesother than those listed for a polypeptide in Table 2, 3, 4, 5, 6, 7, 8,or 9. Preferably, when the candidate polypeptide is being compared to apolypeptide in Table 2, 3, 4, or 5, the candidate polypeptide isobtained from an S. enterica serovar Newport, an S. enterica serovarEnteritidis, an S. enterica serovar Typhimurium, or an S. entericaserovar Dublin, respectively. Preferably, when the candidate polypeptideis being compared to a polypeptide in Table 6, 7, 8, or 9, the candidatepolypeptide is obtained from an E. coli. A candidate polypeptide can beobtained by growth of a microbe under low metal conditions and thesubsequent isolation of a polypeptide by the processes described herein.

It is well known in the art that modifications of amino acids can beaccidentally introduced during sample handling, such as oxidation, andformation of carbamidomethyl derivatives. Further, these types ofmodifications alter the m/z value of a polypeptide fragment. Forinstance, if a polypeptide fragment contains a methoinine that isoxidized the m/z value will be increased by 16 relative to the samefragment that does not contain the oxidized methionine. It is understoodthat the polypeptide fragments of Tables 2, 3, 4, 5, 6, 7, 8, and 9 canbe modified during sample handling.

TABLE 2 Characteristics of polypeptides obtainedfrom Salmonella enterica serovar Newport. mass of approximatepolypeptide molecular fragments predicted amino weight in resultingacid sequence of polypeptide kilodaltons from trypsin the polypeptidedesignation (kDa)¹ digest² fragment Lw221 82 628.39 IEVLR (SEQ ID NO: 1)643.37 QIDIR (SEQ ID NO: 2) 771.42 DINGVVR (SEQ ID NO: 3) 830.45 DVSEIIR(SEQ ID NO: 4) 872.46 LGWRGER (SEQ ID NO: 5) 990.55 EIGLEFKR(SEQ ID NO: 6) 1082.61 IEAGTVPLQR (SEQ ID NO: 7) 1207.58 TGSYADTLPAGR(SEQ ID NO: 8) 1324.75 NKIEAGTVPLQR (SEQ ID NO: 9) 1377.66 TDVYQWENVPK(SEQ ID NO: 10) 1463.74 LYGNLDKTQADAR (SEQ ID NO: 15) 1499.71GDTAWVPPEMIER (SEQ ID NO: 11) 1618.77 TMPGVNLTGNSTSGQR (SEQ ID NO: 13)1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 14) 1727.83 TNFSLNGPLGGDFSFR(SEQ ID NO: 16) 1871.88 DTRGDTAWVPPEMIER (SEQ ID NO: 17) 1980.96WDFAPLQSLELEAGYSR (SEQ ID NO: 18) 1997.06 GMGPENTLILIDGKPVTSR(SEQ ID NO: 19) 2192.94 DTQTGAYMAGAGAYTYNEPGR (SEQ ID NO: 21) 2331.05KGGSEWHGSWNTYFNAPEHK (SEQ ID NO: 22) Lw223A 80 848.45 LYGNLNR(SEQ ID NO: 23) 918.45 LGFYYEK (SEQ ID NO: 24) 1040.60 IVAGDQIIGR(SEQ ID NO: 25) 1097.62 QQPGVSIITR (SEQ ID NO: 26) 1309.63 ITNDQTFTTNR(SEQ ID NO: 27) 1335.71 NPPVNDLADIIR (SEQ ID NO: 28) 1341.66DSNIAGIPGSAANR (SEQ ID NO: 29) 1364.60 SAEGANTYNEPGR (SEQ ID NO: 30)1528.70 GDTNWVPPEMVER (SEQ ID NO: 31) 1564.76 SASGAYVLQWQNGGK(SEQ ID NO: 32) 1735.86 GNFSLSGPLAGDTLTMR (SEQ ID NO: 33) 1750.86EGVTNKDINSVFSWR (SEQ ID NO: 34) 1754.83 MTPQQILDFEAGYSR (SEQ ID NO: 35)1845.91 APNLYQTSEGYLLYSK (SEQ ID NO: 36) 1911.98 ALIEGIEASMAVPLMPDR(SEQ ID NO: 37) 1929.04 GPAAARYGSGAAGGVVNIITK (SEQ ID NO: 38) 1935.01DDIQKNPPVNDLADIIR (SEQ ID NO: 39) 2030.93 QNYGLTHNGIWDWGQSR(SEQ ID NO: 40) 2416.14 RPTNDWHGSLSLYTNQPESSK (SEQ ID NO: 41) 2587.35IVAGDQIIGRSASGAYVLQWQNGGK (SEQ ID NO: 42) 2701.36SEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 43) 2909.36FDYLSESGSNFSPSLNLSQELGEYVK (SEQ ID NO: 44) 2943.50NKSEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 45) Lw223B 74 605.33 NAVFR(SEQ ID NO: 46) 616.37 VPVFR (SEQ ID NO: 47) 808.41 IEGFTSR(SEQ ID NO: 48) 1063.48 YFMAVDYR (SEQ ID NO: 49) 1158.55 QNYALSHNGR(SEQ ID NO: 50) 1210.55 YFMAVDYRF (SEQ ID NO: 51) 1314.62 LSLNYTYNDGR(SEQ ID NO: 52) 1329.77 IFEPLALTTGIR (SEQ ID NO: 53) 1345.55 DDYGYTEDGRR(SEQ ID NO: 54) 1526.73 EVPGVQLTNEGDNR (SEQ ID NO: 55) 1649.90GLDSSYTLILIDGKR (SEQ ID NO: 56) 1677.74 DEQQSSATTATGETPR (SEQ ID NO: 57)1740.90 DAPASISVITQQDLQR (SEQ ID NO: 58) 1744.69 MDDHETYGDHWSPR(SEQ ID NO: 59) 1750.84 WHGSVTVDSTIQEHR (SEQ ID NO: 60) 1792.88GEEGILEGVEASVTTFR (SEQ ID NO: 61) 1814.85 TSASQYALFLEDEWR(SEQ ID NO: 62) 1906.92 TPGGYVVWDTGAAWQATK (SEQ ID NO: 63) 1934.88EKDEQQSSATTATGETPR (SEQ ID NO: 64) 1952.94 HNDFDLNWIPVDAIER(SEQ ID NO: 65) 1987.04 IQGVETELKVPFNEAWK (SEQ ID NO: 66) 2242.03TPDVNAAPGYSNFVGFETNSR (SEQ ID NO: 67) 2538.26 IVGSPDLKPETSESWELGLYYR(SEQ ID NO: 68) 2587.24 DRGDTYNGQFFTSGPLIDGVLGMK (SEQ ID NO: 69) 2710.17DGNVEFAWTPNENHDVTAGYGFDR (SEQ ID NO: 70) P4 65 1303.65 WQSTSVNDVLR(SEQ ID NO: 71) 1398.57 YDSDYSAYPYR (SEQ ID NO: 72) 1508.73TLYGALEHTFSDR (SEQ ID NO: 73) 1792.85 QWEGAFEGLTAGVSWR (SEQ ID NO: 74)1868.85 QTTTPGTGYVPEGYDQR (SEQ ID NO: 75) 1932.87 TDYDAYYSPGSPLIDTR(SEQ ID NO: 76) 2023.92 HGTWQTSAGWEFIEGYR (SEQ ID NO: 77) 2086.08LPGVDIAQSGGAGQNSSIFIR (SEQ ID NO: 78) 2257.21 LNLAGVSGSADLSQFPVSLVQR(SEQ ID NO: 79) Lw224 56 1100.52 DDAAGQAIANR (SEQ ID NO: 80) 1131.59SQSALGTAIER (SEQ ID NO: 81) 1254.69 IDAALAQVDALR (SEQ ID NO: 82) 1715.73IEDSDYATEVSNMSR (SEQ ID NO: 83) 1756.93 QINSQTLGLDSLNVQK (SEQ ID NO: 84)1958.86 SRIEDSDYATEVSNMSR (SEQ ID NO: 85) 2034.01 FNSAITNLGNTVNNLSEAR(SEQ ID NO: 86) 2669.30 NANDGISIAQTTEGALNEINNNLQR (SEQ ID NO: 87)2804.34 ELAVQSANSTNSQSDLDSIQAEITQR (SEQ ID NO: 88) 2859.59AQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 89) 3059.51VRELAVQSANSTNSQSDLDSIQAEITQR (SEQ ID NO: 90) Lw225 55 958.48 SDLGAVQNR(SEQ ID NO: 91) 1100.52 DDAAGQAIANR (SEQ ID NO: 92) 1131.59 SQSALGTAIER(SEQ ID NO: 93) 1143.59 TALNQLGGADGK (SEQ ID NO: 94) 1254.69IDAALAQVDALR (SEQ ID NO: 95) 1603.77 ADVDADGNVSLATGATK (SEQ ID NO: 96)1613.81 INSAKDDAAGQAIANR (SEQ ID NO: 97) 1621.82 AGITGTTTETGSVKDGK(SEQ ID NO: 98) 1639.79 YDVDSTGVTQSLDLK (SEQ ID NO: 99) 1708.75NYYVEVDFTDTTDK (SEQ ID NO: 100) 1715.73 IEDSDYATEVSNMSR (SEQ ID NO: 101)1770.95 QINSQTLGLDTLNVQK (SEQ ID NO: 102) 1903.98 LNEIDRVSGQTQFNGVK(SEQ ID NO: 103) 1958.86 SRIEDSDYATEVSNMSR (SEQ ID NO: 104) 2084.12AQVINTNSLSLLTQNNLNK (SEQ ID NO: 105) 2195.17 IDAALAQVDALRSDLGAVQNR(SEQ ID NO: 106) 2669.30 NANDGISIAQTTEGALNEINNNLQR (SEQ ID NO: 107)2804.34 ELAVQSANSTNSQSDLDSIQAEITQR (SEQ ID NO: 108) 3059.51VRELAVQSANSTNSQSDLDSIQAEITQR (SEQ ID NO: 109) Lw226 52 787.46 SVVQTVR(SEQ ID NO: 110) 799.43 QQLANAR (SEQ ID NO: 111) 801.43 LSQDLAR(SEQ ID NO: 112) 827.45 LSNPELR (SEQ ID NO: 113) 913.53 NLSLLQAR(SEQ ID NO: 114) 1089.50 ANSNNGNPFR (SEQ ID NO: 115) 1285.63 NNLDNAVEELR(SEQ ID NO: 116) 1381.76 YTYLINQLNIK (SEQ ID NO: 117) 1549.77AQYDTVLANEVTAR (SEQ ID NO: 118) 1615.87 FNVGLVAITDVQNAR (SEQ ID NO: 119)1661.90 VLNAIDVLSYTQAQK (SEQ ID NO: 120) 1737.90 TIVDVLDATTTLYDAK(SEQ ID NO: 121) 1828.90 SSFNNINASISSINAYK (SEQ ID NO: 122) 2033.96QAQYNFVGASEQLESAHR (SEQ ID NO: 123) 2184.09 SPLLPQLGLGADYTYSNGYR(SEQ ID NO: 124) 2208.09 QVTGNYYPELASLNVEHFK (SEQ ID NO: 125) 2226.06QAVVSAQSSLDAMEAGYSVGTR (SEQ ID NO: 126) 2684.26DANGINSNETSASLQLTQTLFDMSK (SEQ ID NO: 127) 2748.32QAQDGHLPTLNLTASTGISDTSYSGSK (SEQ ID NO: 128) 2886.44AAGIQDVTYQTDQQTLILNTANAYFK (SEQ ID NO: 129) Lw227 45 665.35 WGYIK(SEQ ID NO: 130) 730.43 LSLAATR (SEQ ID NO: 131) 812.44 IFATYAK(SEQ ID NO: 132) 858.46 LGQEVWK (SEQ ID NO: 133) 963.46 VDFHGYAR(SEQ ID NO: 134) 1150.53 DTANDVFDVR (SEQ ID NO: 135) 1223.60 WDEKWGYIK(SEQ ID NO: 136) 1411.67 YAAATNSGISTNSR (SEQ ID NO: 137) 1422.69WGYIKDGDNISR (SEQ ID NO: 138) 1658.80 FVVQYATDAMTTQGK (SEQ ID NO: 139)1683.90 NLIEWLPGSTIWAGK (SEQ ID NO: 140) 1780.79 DGWMFTAEHTQSMLK(SEQ ID NO: 141) 1964.99 WTPIMSTLLEVGYDNVK (SEQ ID NO: 142) 2086.07LAGLQTNPDGVLELGVDYGR (SEQ ID NO: 143) 2181.97 STEAGGSYTFSSQNIYDEVK(SEQ ID NO: 144) 2296.21 ITLAQQWQAGDSIWSRPAIR (SEQ ID NO: 145) 3100.35SFYFDTNVAYSVNQQNDWESTDPAFR (SEQ ID NO: 146) 3314.49STEAGGSYTFSSQNIYDEVKDTANDVFDVR (SEQ ID NO: 147) Lw228A 38 718.44 LAFAGLK(SEQ ID NO: 148) 867.44 TTGVATYR (SEQ ID NO: 149) 1057.56 NAEVWAAGLK(SEQ ID NO: 150) 1103.50 NMSTFVDYK (SEQ ID NO: 151) 1121.57 DGNKLDLYGK(SEQ ID NO: 152) 1296.54 FADYGSFDYGR (SEQ ID NO: 153) 1638.83VSTDNIVAVGLNYQF (SEQ ID NO: 154) 2218.07 NTDFFGLVEGLNFAAQYQGK(SEQ ID NO: 155) 2382.03 VHAQHYFSDDNGSDGDKTYAR (SEQ ID NO: 156) 2389.09GETQINDQLTGFGQWEYEFK (SEQ ID NO: )157 2603.24 NTDFFGLVEGLNFAAQYQGKNDR(SEQ ID NO: 158) 2716.25 GETQINDQLTGFGQWEYEFKGNR (SEQ ID NO: 159)2757.28 NLGTYGDQDLVEYIDVGATYYFNK (SEQ ID NO: 160) 2805.41TQNFEAVAQYQFDFGLRPSIAYLK (SEQ ID NO: 161) 2834.36LGFKGETQINDQLTGFGQWEYEFK (SEQ ID NO: 162) 3065.32DGAYESNGDGFGLSATYEYEGFGVGAAYAK (SEQ ID NO: 163) 3450.49NDRDGAYESNGDGFGLSATYEYEGFGVGAAYAK (SEQ ID NO: 164) Lw228B 38 704.42VAFAGLK (SEQ ID NO: 165) 793.37 LYGNGDR (SEQ ID NO: 166) 900.41 GNGYATYR(SEQ ID NO: 167) 908.50 ATVYTGGLK (SEQ ID NO: 168) 1105.58 DGNKLDLFGK(SEQ ID NO: 169) 1204.51 FADAGSFDYGR (SEQ ID NO: 171) 1800.82DISNGYGASYGDQDIVK (SEQ ID NO: 174) 1834.81 FGTSNGSNPSTSYGFANK(SEQ ID NO: 175) 1944.95 LDLFGKVDGLNYFSDDK (SEQ ID NO: 170) 1985.93GKDISNGYGASYGDQDIVK (SEQ ID NO: 176) 2247.08 NTDFFGLVDGLDFALQYQGK(SEQ ID NO: 177) 2382.01 VDGLNYFSDDKGSDGDQTYMR (SEQ ID NO: 178) 3004.51AQNFEVVAQYQFDFGLRPSVAYLQSK (SEQ ID NO: 180) 3133.52SLLNQNGDGYGGSLTYAIGEGFSVGGAITTSK (SEQ ID NO: 181) Lw230A 36 817.43LGGMVWR (SEQ ID NO: 182) 871.51 RVEIEVK (SEQ ID NO: 183) 914.52AQGVQLTAK (SEQ ID NO: 184) 1024.46 DNTWYAGAK (SEQ ID NO: 185) 1082.54SDVLFNFNK (SEQ ID NO: 186) 1156.59 AALIDCLAPDR (SEQ ID NO: 187) 1263.65DGSVVVLGFTDR (SEQ ID NO: 188) 1377.76 RAQSVVDYLISK (SEQ ID NO: 189)1380.65 IGSDAYNQGLSEK (SEQ ID NO: 190) 1536.75 IGSDAYNQGLSEKR(SEQ ID NO: 191) 1639.81 LGYPITDDLDVYTR (SEQ ID NO: 192) 2302.20FGQQEAAPVVAPAPAPAPEVQTK (SEQ ID NO: 193) 2615.29DHDTGVSPVFAGGIEYAITPEIATR (SEQ ID NO: 194) 2672.37STLKPEGQQALDQLYSQLSNLDPK (SEQ ID NO: 195) 3422.69LEYQWTNNIGDANTIGTRPDNGLLSVGVSYR (SEQ ID NO: 196) Lw233 22 1050.53VRPYVGVGVNYTTFFDNDFNDNGK (SEQ ID NO: 202) 1221.57NAGLSDLSFKDSWGAAGQVGVDYLINR (SEQ ID NO: 203) 1587.75VGTGATGDIATVHLLPPTLMAQWYFGDSSSK (SEQ ID NO: 204) 1734.82 MSGFNLK(SEQ ID NO: 205) 1819.89 FQTTDYPTYK (SEQ ID NO: 206) 2737.28GQYYGITAGPAYR (SEQ ID NO: 207) 2852.41 SVDVGTWIAGVGYR (SEQ ID NO: 208)3219.59 SVDVGTWIAGVGYRF (SEQ ID NO: 209) Lw234 18 795.39LNDWASIYGVVGVGYGK (SEQ ID NO: 210) 1262.58 YEQDDNPLGVIGSFTYTEK(SEQ ID NO: 211) 1415.68 YEQDDNPLGVIGSFTYTEKDR (SEQ ID NO: 212) 1478.75YRYEQDDNPLGVIGSFTYTEK (SEQ ID NO: 213) 1625.82 LDNQATK (SEQ ID NO: 214)1796.91 SDVQAAKDDAAR (SEQ ID NO: 215) 2175.00 ANQRLDNQATK(SEQ ID NO: 216) 2446.13 VDQLSNDVNAMR (SEQ ID NO: 217) 2494.17IDQLSSDVQTLNAK (SEQ ID NO: 218) Lw235 12 788.40 VDQLSNDVNAMRSDVQAAK(SEQ ID NO: 219) 1245.59 IDQLSSDVQTLNAKVDQLSNDVNAMR (SEQ ID NO: 220)¹Molecular weight as determined by SDS-PAGE. ²The mass of a polypeptidefragment can be converted to m/z value by adding 1 to the mass. Eachmass includes a range of plus or minus 300 parts per million (ppm).

TABLE 3 Characteristics of polypeptides obtainedfrom S. enteritidis serovar Enteritidis. mass of approximate polypeptidemolecular fragments predicted amino weight in resulting acid sequence ofpolypeptide kilodaltons from trypsin the polypeptide designation (kDa)¹digest² fragment Lw 98 92 728.43 WVVLGR (SEQ ID NO: 221) 815.38 SYYLDR(SEQ ID NO: 222) 888.41 YGYAYPR (SEQ ID NO: 223) 957.50 AGLGYVHNK(SEQ ID NO: 224) 972.50 QNLEASGVR (SEQ ID NO: 225) 986.54 LAGDLETLR(SEQ ID NO: 226) 998.45 GYFPTDGSR (SEQ ID NO: 227) 1008.47 WGYGDGLGGK(SEQ ID NO: 228) 1047.53 GFQSNTIGPK (SEQ ID NO: 229) 1076.59 LVFQEGVSAK(SEQ ID NO: 230) 1113.56 DIHFEGLQR (SEQ ID NO: 231) 1180.59 AEQFQFNIGK(SEQ ID NO: 232) 1219.61 IEPGELYNGTK (SEQ ID NO: 233) 1276.60GLEDFYYSVGK (SEQ ID NO: 234) 1282.74 VAVGAALLSMPVR (SEQ ID NO: 235)1338.66 ALFATGNFEDVR (SEQ ID NO: 236) 1384.65 VTIPGSDNEYYK(SEQ ID NO: 237) 1401.71 VPGSPDQVDVVYK (SEQ ID NO: 238) 1402.67LSNMQPQIAMDR (SEQ ID NO: 239) 1470.69 DEVPWWNVVGDR (SEQ ID NO: 240)1519.80 ERPTIASITFSGNK (SEQ ID NO: 241) 1527.72 LGFFETVDTDTQR(SEQ ID NO: 242) 1648.75 TGDTVNDEDISNTIR (SEQ ID NO: 243) 1691.87FNIDSTQVSLTPDKK (SEQ ID NO: 244) 1712.86 GIYITVNITEGDQYK(SEQ ID NO: 1352) 1732.81 QMEGAWLGSDLVDQGK (SEQ ID NO: 1353) 1758.82YDGDKAEQFQFNIGK (SEQ ID NO: 1354) 1786.87 VSLDTATYVPIDNDHK(SEQ ID NO: 1355) 1894.97 LSGVQVSGNLAGHSAEIEK (SEQ ID NO: 1356) 1953.86EMPFYENFYAGGSSTVR (SEQ ID NO: 1357) 2159.05 SYGTDVTLGFPINEYNTLR(SEQ ID NO: 1358) 2254.95 IFYNDFEADDADLSDYTNK (SEQ ID NO: 1359) 2284.27LLIASLLFSSATVYGAEGFVVK (SEQ ID NO: 1360) 2793.46IQQINIVGNHAFSTEELISHFQLR (SEQ ID NO: 1361) 2881.34NDYQTYSELSVTNPYFTVDGVSLGGR (SEQ ID NO: 1362) Lw 99 91 904.51 LVQLNYR(SEQ ID NO: 1363) 924.43 SGVQYDTR (SEQ ID NO: 1364) 945.53 SGFLIPNAK(SEQ ID NO: 1365) 1004.49 WGSLNTEAK (SEQ ID NO: 1366) 1050.50 IYDDAAVER(SEQ ID NO: 197) 1075.60 VDGKLIFER (SEQ ID NO: 198) 1109.55 QAEGQPEPVR(SEQ ID NO: 199) 1199.63 VQYLYVPYR (SEQ ID NO: 200) 1276.56 YGSSTDGYATQK(SEQ ID NO: 201) 1294.58 GNIMWENEFR (SEQ ID NO: 245) 1307.68 LQADEVQLHQK(SEQ ID NO: 246) 1343.65 EEQVAEIWNAR (SEQ ID NO: 247) 1375.72IYGQAVHFVNTK (SEQ ID NO: 248) 1417.75 IASANQVTTGVTTR (SEQ ID NO: 249)1450.68 RGNIMWENEFR (SEQ ID NO: 250) 1509.63 VSDSSYFNDFDSK(SEQ ID NO: 251) 1510.72 TGSLVWAGDTYWR (SEQ ID NO: 252) 1601.89VHLEPTINLPLSNR (SEQ ID NO: 253) 1618.81 EEQVAEIWNARFK (SEQ ID NO: 254)1624.79 GLSSNYGLGTQEMLR (SEQ ID NO: 255) 1668.72 DTNVWEGDYQMVGR(SEQ ID NO: 256) 1766.91 NGINQVGAVASWPIADR (SEQ ID NO: 257) 1766.91NGINQVGAVASWPIADR (SEQ ID NO: 258) 1792.85 DMAMLAPGYTQTLEPR(SEQ ID NO: 259) 1808.87 FNVSVGQIYYFTESR (SEQ ID NO: 260) 1832.89FSVGYAVQNFDATVSTK (SEQ ID NO: 261) 2013.02 TVDALGNVHYDDNQVILK(SEQ ID NO: 262) 2088.13 VGPVPIFYSPYLQLPVGDK (SEQ ID NO: 263) 2269.01GNYPDDAVFTGNVDIMQGNSR (SEQ ID NO: 264) 2298.03 WENDDKTGSLVWAGDTYWR(SEQ ID NO: 265) 2453.09 LMATHYQQTNLDSYNSDPNNK (SEQ ID NO: 266) 2554.16DQSGIYNYDSSLLQSDYNGLFR (SEQ ID NO: 267) 2572.20 YASPEYIQATLPSYYSTAEQYK(SEQ ID NO: 268) Lw 101 86 643.37 QIDIR (SEQ ID NO: 269) 872.46 LGWRGER(SEQ ID NO: 270) 950.49 ETNRLYR (SEQ ID NO: 271) 990.55 EIGLEFKR(SEQ ID NO: 272) 1082.61 IEAGTVPLQR (SEQ ID NO: 273) 1084.57 GNNRQIDIR(SEQ ID NO: 274) 1095.49 DNYGKETNR (SEQ ID NO: 275) 1151.68 IEVLRGPAAAR(SEQ ID NO: 276) 1181.55 NINQGHQSER (SEQ ID NO: 277) 1207.58TGSYADTLPAGR (SEQ ID NO: 278) 1324.75 NKIEAGTVPLQR (SEQ ID NO: 279)1365.74 NPPARDVSEIIR (SEQ ID NO: 280) 1377.66 TDVYQWENVPK(SEQ ID NO: 281) 1411.78 EGVINKDINGVVR (SEQ ID NO: 282) 1432.77YGNGAAGGVVNIITK (SEQ ID NO: 283) 1463.74 LYGNLDKTQADAR (SEQ ID NO: 284)1499.71 GDTAWVPPEMIER (SEQ ID NO: 285) 1560.86 YGNGAAGGVVNIITKK(SEQ ID NO: 286) 1560.86 YGNGAAGGVVNIITKK (SEQ ID NO: 287) 1584.74KYDYQGNPVTGTDK (SEQ ID NO: 288) 1618.77 TMPGVNLTGNSTSGQR(SEQ ID NO: 289) 1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 290) 1633.84NVSLTGGVDNLFDKR (SEQ ID NO: 291) 1727.83 TNFSLNGPLGGDFSFR(SEQ ID NO: 292) 1870.95 APSLYQTNPNYILYSK (SEQ ID NO: 293) 1903.94INNGKTDVYQWENVPK (SEQ ID NO: 294) 1974.95 NSRMPEGLAGGTEGIFDPK(SEQ ID NO: 295) 1980.96 WDFAPLQSLELEAGYSR (SEQ ID NO: 296) 1997.06GMGPENTLILIDGKPVTSR (SEQ ID NO: 297) 2078.00 QGNLYAGDTQNTNTNQLVK(SEQ ID NO: 298) 2192.94 DTQTGAYMAGAGAYTYNEPGR (SEQ ID NO: 299) 2233.14AYKAPSLYQTNPNYILYSK (SEQ ID NO: 300) 2371.13 NINQGHQSERTGSYADTLPAGR(SEQ ID NO: 301) 2531.24 TNFSLNGPLGGDFSFRLYGNLDK (SEQ ID NO: 302)2622.42 QIDIRGMGPENTLILIDGKPVTSR (SEQ ID NO: 303) 2632.20MKDQLSNSQTFMGGNIPGYSSTNR (SEQ ID NO: 304) 3098.47FDHHSIVGDNWSPSLNLSQGLGDDFTLK (SEQ ID NO: 305) 3211.45QNYSLTWNGGWNNGVTTSNWVQYEHTR (SEQ ID NO: 306) 3473.51DTQTGAYMAGAGAYTYNEPGRTWYMSINTHF (SEQ ID NO: 307) Lw 102 83 610.29 YSWR(SEQ ID NO: 308) 628.39 IEVIR (SEQ ID NO: 309) 848.45 LYGNLNR(SEQ ID NO: 310) 918.45 LGFYYEK (SEQ ID NO: 311) 1040.60 IVAGDQIIGR(SEQ ID NO: 312) 1097.62 QQPGVSIITR (SEQ ID NO: 313) 1141.60 NLDPEISINK(SEQ ID NO: 314) 1153.63 DTGNPLSIIPK (SEQ ID NO: 315) 1162.50MNEGLSGGGEGR (SEQ ID NO: 316) 1218.66 LNVGISNIFDK (SEQ ID NO: 317)1309.63 ITNDQTFTTNR (SEQ ID NO: 318) 1335.71 NPPVNDLADIIR(SEQ ID NO: 319) 1341.66 DSNIAGIPGSAANR (SEQ ID NO: 320) 1364.60SAEGANTYNEPGR (SEQ ID NO: 321) 1405.76 YGSGAAGGVVNIITK (SEQ ID NO: 322)1460.70 MPGVNLTGNSASGTR (SEQ ID NO: 323) 1528.70 GDTNWVPPEMVER(SEQ ID NO: 324) 1564.76 SASGAYVLQWQNGGK (SEQ ID NO: 325) 1564.76SASGAYVLQWQNGGK (SEQ ID NO: 326) 1735.86 GNFSLSGPLAGDTLTMR(SEQ ID NO: 327) 1750.86 EGVTNKDINSVFSWR (SEQ ID NO: 328) 1754.83MTPQQILDFEAGYSR (SEQ ID NO: 329) 1845.91 APNLYQTSEGYLLYSK(SEQ ID NO: 330) 1880.96 ALGAYSLVGANVNYDINK (SEQ ID NO: 331) 1911.98ALIEGIEASMAVPLMPDR (SEQ ID NO: 332) 1954.02 GMGPENTLVLIDGVPVTSR(SEQ ID NO: 333) 2030.93 QNYGLTHNGIWDWGQSR (SEQ ID NO: 334) 2261.06EIGLEFTVDDYHASVTYFR (SEQ ID NO: 335) 2397.12 LYGNLNRTDADSWDINSSAGTK(SEQ ID NO: 336) 2416.14 RPTNDWHGSLSLYTNQPESSK (SEQ ID NO: 337) 2701.36SEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 338) 2909.36FDYLSESGSNFSPSLNLSQELGEYVK (SEQ ID NO: 339) 2943.50NKSEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 340) Lw 103 78 605.33 NAVFR(SEQ ID NO: 341) 614.38 IEVVR (SEQ ID NO: 342) 616.37 VPVFR(SEQ ID NO: 343) 808.41 IEGFTSR (SEQ ID NO: 344) 836.42 GGWATAFK(SEQ ID NO: 345) 989.50 VPFNEAWK (SEQ ID NO: 346) 1060.52 WDLGNSELK(SEQ ID NO: 347) 1063.48 YFMAVDYR (SEQ ID NO: 348) 1140.66 LRAGVLNVGDK(SEQ ID NO: 349) 1158.55 QNYALSHNGR (SEQ ID NO: 350) 1177.52 QDRDSDSLDK(SEQ ID NO: 351) 1177.52 QDRDSDSLDK (SEQ ID NO: 352) 1210.55 YFMAVDYRF(SEQ ID NO: 353) 1314.62 LSLNYTYNDGR (SEQ ID NO: 354) 1329.77IFEPLALTTGIR (SEQ ID NO: 355) 1345.55 DDYGYTEDGRR (SEQ ID NO: 356)1493.80 GLDSSYTLILIDGK (SEQ ID NO: 357) 1526.73 EVPGVQLTNEGDNR(SEQ ID NO: 358) 1570.82 AYLVYNATDTLTVK (SEQ ID NO: 359) 1649.90GLDSSYTLILIDGKR (SEQ ID NO: 360) 1654.83 EVPGVQLTNEGDNRK(SEQ ID NO: 361) 1740.90 DAPASISVITQQDLQR (SEQ ID NO: 362) 1744.69MDDHETYGDHWSPR (SEQ ID NO: 363) 1750.84 WHGSVTVDSTIQEHR (SEQ ID NO: 364)1792.88 GEEGILEGVEASVTTFR (SEQ ID NO: 365) 1814.85 TSASQYALFLEDEWR(SEQ ID NO: 366) 1906.92 TPGGYVVWDTGAAWQATK (SEQ ID NO: 367) 1952.94HNDFDLNWIPVDAIER (SEQ ID NO: 368) 2242.03 TPDVNAAPGYSNFVGFETNSR(SEQ ID NO: 369) 2538.26 IVGSPDLKPETSESWELGLYYR (SEQ ID NO: 370) 2710.17DGNVEFAWTPNENHDVTAGYGF (SEQ ID NO: 371) Lw 104 55 787.46 SVVQTVR(SEQ ID NO: 372) 801.43 LSQDLAR (SEQ ID NO: 373) 913.53 NLSLLQAR(SEQ ID NO: 374) 1179.55 SAADRDAAFEK (SEQ ID NO: 375) 1226.55ANSNNGNPFRH (SEQ ID NO: 376) 1226.55 ANSNNGNPFRH (SEQ ID NO: 377)1285.63 NNLDNAVEELR (SEQ ID NO: 378) 1381.76 YTYLINQLNIK(SEQ ID NO: 379) 1549.77 AQYDTVLANEVTAR (SEQ ID NO: 380) 1615.87FNVGLVAITDVQNAR (SEQ ID NO: 381) 1661.90 VLNAIDVLSYTQAQK(SEQ ID NO: 382) 1737.90 TIVDVLDATTTLYDAK (SEQ ID NO: 383) 1828.90SSFNNINASISSINAYK (SEQ ID NO: 384) 2033.96 QAQYNFVGASEQLESAHR(SEQ ID NO: 385) 2184.09 SPLLPQLGLGADYTYSNGYR (SEQ ID NO: 386) 2208.09QVTGNYYPELASLNVEHFK (SEQ ID NO: 387) 2226.06 QAVVSAQSSLDAMEAGYSVGTR(SEQ ID NO: 388) 2748.32 QAQDGHLPTLNLTASTGISDTSYSGSK (SEQ ID NO: 389)2886.44 AAGIQDVTYQTDQQTLILNTANAYFK (SEQ ID NO: 390) Lw 106A 40 691.39LDLFGK (SEQ ID NO: 391) 704.42 VAFAGLK (SEQ ID NO: 392) 900.41 GNGYATYR(SEQ ID NO: 393) 908.50 ATVYTGGLK (SEQ ID NO: 394) 973.45 STSYGFANK(SEQ ID NO: 395) 1105.58 DGNKLDLFGK (SEQ ID NO: 396) 1128.45 GSDGDQTYMR(SEQ ID NO: 397) 1190.53 NGSVSGENTNGR (SEQ ID NO: 398) 1204.51FADAGSFDYGR (SEQ ID NO: 399) 1438.68 YVDVGATYYFNK (SEQ ID NO: 400)1800.82 DISNGYGASYGDQDIVK (SEQ ID NO: 401) 1890.93 VAFAGLKFADAGSFDYGR(SEQ ID NO: 402) 1989.84 TADQNNTADEHLYGNGDR (SEQ ID NO: 403) 2247.08NTDFFGLVDGLDFALQYQGK (SEQ ID NO: 404) 2339.08 YDANNIYLAAQYSQTYNATR(SEQ ID NO: 405) 2405.02 VDGLHYFSDDKGSDGDQTYMR (SEQ ID NO: 406) 3004.51AQNFEVVAQYQFDFGLRPSVAYL (SEQ ID NO: 407) Lw 106B 39 718.44 LAFAGLK(SEQ ID NO: 408) 867.44 TTGVATYR (SEQ ID NO: 409) 1057.56 NAEVWAAGLK(SEQ ID NO: 410) 1103.50 NMSTFVDYK (SEQ ID NO: 411) 1121.57 DGNKLDLYGK(SEQ ID NO: 412) 1279.63 INLLDDSDFTK (SEQ ID NO: 413) 1296.54FADYGSFDYGR (SEQ ID NO: 414) 1638.83 VSTDNIVAVGLNYQF (SEQ ID NO: 415)1890.78 VHAQHYFSDDNGSDGDK (SEQ ID NO: 416) 2218.07 NTDFFGLVEGLNFAAQYQGK(SEQ ID NO: 417) 2218.07 NTDFFGLVEGLNFAAQYQGK (SEQ ID NO: 418) 2382.03VHAQHYFSDDNGSDGDKTYAR (SEQ ID NO: 419) 2389.09 GETQINDQLTGFGQWEYEFK(SEQ ID NO: 420) 2757.28 NLGTYGDQDLVEYIDVGATYYFNK (SEQ ID NO: 421)2805.41 TQNFEAVAQYQFDFGLRPSIAYLK (SEQ ID NO: 422) 3067.50TTGVATYRNTDFFGLVEGLNFAAQYQGK (SEQ ID NO: 423) Lw 108 38 817.43 LGGMVWR(SEQ ID NO: 424) 1024.46 DNTWYAGAK (SEQ ID NO: 425) 1082.54 SDVLFNFNK(SEQ ID NO: 426) 1156.59 AALIDCLAPDR (SEQ ID NO: 427) 1221.66AQSVVDYLISK (SEQ ID NO: 428) 1232.63 LGGMVWRADTK (SEQ ID NO: 429)1263.65 DGSVVVLGFTDR (SEQ ID NO: 430) 1377.76 RAQSVVDYLISK(SEQ ID NO: 431) 1380.65 IGSDAYNQGLSEK (SEQ ID NO: 432) 1469.70MPYKGDNINGAYK (SEQ ID NO: 433) 1536.75 IGSDAYNQGLSEKR (SEQ ID NO: 434)1639.81 LGYPITDDLDVYTR (SEQ ID NO: 435) 2302.20 FGQQEAAPVVAPAPAPAPEVQTK(SEQ ID NO: 436) 2615.29 DHDTGVSPVFAGGIEYAITPEIATR (SEQ ID NO: 437)2672.37 STLKPEGQQALDQLYSQLSNLDPK (SEQ ID NO: 438) 3422.69LEYQWTNNIGDANTIGTRPDNGLLSVGVSYR (SEQ ID NO: 439) ¹Molecular weight asdetermined by SDS-PAGE. ²The mass of a polypeptide fragment can beconverted to m/z value by adding 1 to the mass. Each mass includes arange of plus or minus 1 Dalton.

TABLE 4 Characteristics of polypeptides obtainedfrom S. enteritidis serovar Typhimurium. mass value of approximatepolypeptide molecular fragments predicted amino weight in resultingacid sequence of polypeptide kilodaltons from trypsin the polypeptidedesignation (kDa)¹ digest² fragment Lw 111 86 990.55 EIGLEFKR(SEQ ID NO: 440) 1082.61 IEAGTVPLQR (SEQ ID NO: 441) 1181.55 NINQGHQSER(SEQ ID NO: 442) 1207.58 TGSYADTLPAGR (SEQ ID NO: 443) 1306.65DGWLAGVTWFR (SEQ ID NO: 444) 1324.75 NKIEAGTVPLQR (SEQ ID NO: 445)1377.66 TDVYQWENVPK (SEQ ID NO: 446) 1432.77 YGNGAAGGVVNIITK(SEQ ID NO: 447) 1477.74 NVSLTGGVDNLFDK (SEQ ID NO: 448) 1499.71GDTAWVPPEMIER (SEQ ID NO: 449) 1584.74 KYDYQGNPVTGTDK (SEQ ID NO: 450)1617.77 MPEGLAGGTEGIFDPK (SEQ ID NO: 451) 1618.77 TMPGVNLTGNSTSGQR(SEQ ID NO: 452) 1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 453) 1658.79QDVSLQSTFTWYGK (SEQ ID NO: 454) 1727.83 TNFSLNGPLGGDFSFR(SEQ ID NO: 455) 1870.95 APSLYQTNPNYILYSK (SEQ ID NO: 456) 1980.96WDFAPLQSLELEAGYSR (SEQ ID NO: 457) 1997.06 GMGPENTLILIDGKPVTSR(SEQ ID NO: 458) 2021.05 QAVSPYSIVGLSATWDVTK (SEQ ID NO: 459) 2078.00QGNLYAGDTQNTNTNQLVK (SEQ ID NO: 460) 2118.15 LSIIPQYTLNSTLSWQVR(SEQ ID NO: 461) 2192.94 DTQTGAYMAGAGAYTYNEPGR (SEQ ID NO: 462) 2202.95GGSEWHGSWNTYFNAPEHK (SEQ ID NO: 463) 2331.05 KGGSEWHGSWNTYFNAPEHK(SEQ ID NO: 464) 2373.07 DQLSNSQTFMGGNIPGYSSTNR (SEQ ID NO: 465) 2632.20MKDQLSNSQTFMGGNIPGYSSTNR (SEQ ID NO: 466) 3098.47FDHHSIVGDNWSPSLNLSQGLGDDFTLK (SEQ ID NO: 467) Lw 112 82 610.29 YSWR(SEQ ID NO: 468) 848.45 LYGNLNR (SEQ ID NO: 469) 918.45 LGFYYEK(SEQ ID NO: 470) 1040.60 IVAGDQIIGR (SEQ ID NO: 471) 1094.54 DINSVFSWK(SEQ ID NO: 472) 1097.62 QQPGVSIITR (SEQ ID NO: 473) 1153.63 DTGNPLSIIPK(SEQ ID NO: 474) 1162.50 MNEGLSGGGEGR (SEQ ID NO: 475) 1208.64QKPRTHAESR (SEQ ID NO: 476) 1218.66 LNVGISNIFDK (SEQ ID NO: 477) 1309.63ITNDQTFTTNR (SEQ ID NO: 478) 1335.71 NPPVNDLADIIR (SEQ ID NO: 479)1341.66 DSNIAGIPGSAANR (SEQ ID NO: 480) 1364.60 SAEGANTYNEPGR(SEQ ID NO: 481) 1405.76 YGSGAAGGVVNIITK (SEQ ID NO: 482) 1528.70GDTNWVPPEMVER (SEQ ID NO: 483) 1564.76 SASGAYVLQWQNGGK (SEQ ID NO: 484)1566.68 TDADSWDINSSAGTK (SEQ ID NO: 485) 1735.86 GNFSLSGPLAGDTLTMR(SEQ ID NO: 486) 1754.83 MTPQQILDFEAGYSR (SEQ ID NO: 487) 1845.91APNLYQTSEGYLLYSK (SEQ ID NO: 488) 1880.96 ALGAYSLVGANVNYDINK(SEQ ID NO: 489) 1882.89 LNWNTNATYMIASEQK (SEQ ID NO: 490) 1911.98ALIEGIEASMAVPLMPDR (SEQ ID NO: 491) 1929.95 ITNDQTFTTNRLTSYR(SEQ ID NO: 492) 1954.02 GMGPENTLVLIDGVPVTSR (SEQ ID NO: 493) 2030.93QNYGLTHNGIWDWGQSR (SEQ ID NO: 494) 2192.12 AFKAPNLYQTSEGYLLYSK(SEQ ID NO: 495) 2261.06 EIGLEFTVDDYHASVTYFR (SEQ ID NO: 496) 2416.14RPTNDWHGSLSLYTNQPESSK (SEQ ID NO: 497) 2449.22 DITSGGCYLVGNKNLDPEISINK(SEQ ID NO: 498) 2701.36 SEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 499)2909.36 FDYLSESGSNFSPSLNLSQELGEYVK (SEQ ID NO: 500) 2943.50NKSEISALYVEDNIEPMAGTNIIPGLR (SEQ ID NO: 501) Lw 113 77 957.55 GQRVPVFR(SEQ ID NO: 502) 1158.55 QNYALSHNGR (SEQ ID NO: 503) 1177.52 QDRDSDSLDK(SEQ ID NO: 504) 1210.55 YFMAVDYRF (SEQ ID NO: 505) 1308.79 RPVQNLKDVLK(SEQ ID NO: 506) 1314.62 LSLNYTYNDGR (SEQ ID NO: 507) 1329.77IFEPLALTTGIR (SEQ ID NO: 508) 1345.55 DDYGYTEDGRR (SEQ ID NO: 509)1397.75 VPVFRYYNVNK (SEQ ID NO: 510) 1526.73 EVPGVQLTNEGDNR(SEQ ID NO: 511) 1649.90 GLDSSYTLILIDGKR (SEQ ID NO: 512) 1654.83EVPGVQLTNEGDNRK (SEQ ID NO: 513) 1744.69 MDDHETYGDHWSPR (SEQ ID NO: 514)1750.84 WHGSVTVDSTIQEHR (SEQ ID NO: 515) 1792.88 GEEGILEGVEASVTTFR(SEQ ID NO: 516) 1952.94 HNDFDLNWIPVDAIER (SEQ ID NO: 517) 2021.97WHGSVTVDSTIQEHRDR (SEQ ID NO: 518) 2201.06 QNYALSHNGRWDLGNSELK(SEQ ID NO: 519) 2242.03 TPDVNAAPGYSNFVGFETNSR (SEQ ID NO: 520) Lw 115A40 651.30 NDFTR (SEQ ID NO: 521) 704.42 VAFAGLK (SEQ ID NO: 522) 793.37LYGNGDR (SEQ ID NO: 523) 900.41 GNGYATYR (SEQ ID NO: 524) 908.50ATVYTGGLK (SEQ ID NO: 525) 1105.58 DGNKLDLFGK (SEQ ID NO: 526) 1119.49NMSTYVDYK (SEQ ID NO: 527) 1128.45 GSDGDQTYMR (SEQ ID NO: 528) 1174.53TADQNNTANAR (SEQ ID NO: 529) 1204.51 FADAGSFDYGR (SEQ ID NO: 530)1347.71 INLLDKNDFTR (SEQ ID NO: 531) 1438.68 YVDVGATYYFNK(SEQ ID NO: 532) 1800.82 DISNGYGASYGDQDIVK (SEQ ID NO: 533) 1834.81FGTSNGSNPSTSYGFANK (SEQ ID NO: 534) 1985.93 GKDISNGYGASYGDQDIVK(SEQ ID NO: 535) 2247.08 NTDFFGLVDGLDFALQYQGK (SEQ ID NO: 536) 2339.08YDANNIYLAAQYSQTYNATR (SEQ ID NO: 537) 2405.02 VDGLHYFSDDKGSDGDQTYMR(SEQ ID NO: 538) 3004.51 AQNFEVVAQYQFDFGLRPSVAYLQSK (SEQ ID NO: 539)3133.52 SLLNQNGDGYGGSLTYAIGEGFSVGGAITTSK (SEQ ID NO: 540) Lw 115B 39718.44 LAFAGLK (SEQ ID NO: 541) 867.44 TTGVATYR (SEQ ID NO: 542) 1057.56NAEVWAAGLK (SEQ ID NO: 543) 1103.50 NMSTFVDYK (SEQ ID NO: 544) 1121.57DGNKLDLYGK (SEQ ID NO: 545) 1161.54 GNRTESQGADK (SEQ ID NO: 546) 1279.63INLLDDSDFTK (SEQ ID NO: 547) 1296.54 FADYGSFDYGR (SEQ ID NO: 548)2218.07 NTDFFGLVEGLNFAAQYQGK (SEQ ID NO: 549) 2382.03VHAQHYFSDDNGSDGDKTYAR (SEQ ID NO: 550) 2389.09 GETQINDQLTGFGQWEYEFK(SEQ ID NO: 551) 2757.28 NLGTYGDQDLVEYIDVGATYYFNK (SEQ ID NO: 552)2805.41 TQNFEAVAQYQFDFGLRPSIAYLK (SEQ ID NO: 553) 3450.49NDRDGAYESNGDGFGLSATYEYEGFGVGAAYAK (SEQ ID NO: 554) Lw 117 38 644.36HFTLK (SEQ ID NO: 555) 817.43 LGGMVWR (SEQ ID NO: 556) 871.51 RVEIEVK(SEQ ID NO: 557) 914.52 AQGVQLTAK (SEQ ID NO: 558) 942.48 SNVPGGPSTK(SEQ ID NO: 559) 1024.46 DNTWYAGAK (SEQ ID NO: 560) 1042.58 GIPSDKISAR(SEQ ID NO: 561) 1082.54 SDVLFNFNK (SEQ ID NO: 562) 1140.61 GVKDVVTQPQA(SEQ ID NO: 563) 1221.66 AQSVVDYLISK (SEQ ID NO: 564) 1263.65DGSVVVLGFTDR (SEQ ID NO: 565) 1377.76 RAQSVVDYLISK (SEQ ID NO: 566)1380.65 IGSDAYNQGLSEK (SEQ ID NO: 567) 1469.70 MPYKGDNINGAYK(SEQ ID NO: 568) 1469.70 MPYKGDNINGAYK (SEQ ID NO: 569) 1536.75IGSDAYNQGLSEKR (SEQ ID NO: 570) 1639.81 LGYPITDDLDVYTR (SEQ ID NO: 571)1708.89 HFTLKSDVLFNFNK (SEQ ID NO: 572) 2302.20 FGQQEAAPVVAPAPAPAPEVQTK(SEQ ID NO: 573) 2615.29 DHDTGVSPVFAGGIEYAITPEIATR (SEQ ID NO: 574)2626.29 DGSVVVLGFTDRIGSDAYNQGLSEK (SEQ ID NO: 575) 2672.37STLKPEGQQALDQLYSQLSNLDPK (SEQ ID NO: 576) 3422.69LEYQWTNNIGDANTIGTRPDNGLLSVGVSYR (SEQ ID NO: 577) 3539.75SNVPGGPSTKDHDTGVSPVFAGGIEYAITPEIATR (SEQ ID NO: 578) ¹Molecular weightas determined by SDS-PAGE. ²The mass of a polypeptide fragment can beconverted to m/z value by adding 1 to the mass. Each mass includes arange of plus or minus 1 Dalton.

TABLE 5 Characteristics of polypeptides obtainedfrom S. enteritidis serovar Dublin. mass value of approximatepolypeptide molecular fragments predicted amino weight in resultingacid sequence of polypeptide kilodaltons from trypsin the polypeptidedesignation (kDa)¹ digest² fragment Dublin 1 96 1082.61 IEAGTVPLQR (SD1)(SEQ ID NO: 579) 1207.58 TGSYADTLPAGR (SEQ ID NO: 580) 1298.57TWYMSINTHF (SEQ ID NO: 581) 1377.66 TDVYQWENVPK (SEQ ID NO: 582) 1499.71GDTAWVPPEMIER (SEQ ID NO: 583) 1617.77 MPEGLAGGTEGIFDPK (SEQ ID NO: 585)1727.83 TNFSLNGPLGGDFSFR (SEQ ID NO: 586) 1870.95 APSLYQTNPNYILYSK(SEQ ID NO: 587) 1956.05 GPAAARYGNGAAGGVVNIITK (SEQ ID NO: 588) 1980.96WDFAPLQSLELEAGYSR (SEQ ID NO: 589) 1997.06 GMGPENTLILIDGKPVTSR(SEQ ID NO: 590) 2118.15 LSIIPQYTLNSTLSWQVR (SEQ ID NO: 592) 2192.94DTQTGAYMAGAGAYTYNEPGR (SEQ ID NO: 593) 2202.95 GGSEWHGSWNTYFNAPEHK(SEQ ID NO: 594) 2331.05 KGGSEWHGSWNTYFNAPEHK (SEQ ID NO: 596) Dublin-289 610.29 YSWR (SD2) (SEQ ID NO: 597) 628.39 IEVIR (SEQ ID NO: 598)848.45 LYGNLNR (SEQ ID NO: 599) 918.45 LGFYYEK (SEQ ID NO: 600) 1040.60IVAGDQIIGR (SEQ ID NO: 601) 1097.62 QQPGVSIITR (SEQ ID NO: 602) 1218.66LNVGISNIFDK (SEQ ID NO: 603) 1309.63 ITNDQTFTTNR (SEQ ID NO: 604)1335.71 NPPVNDLADIIR (SEQ ID NO: 605) 1341.66 DSNIAGIPGSAANR(SEQ ID NO: 606) 1364.60 SAEGANTYNEPGR (SEQ ID NO: 607) 1528.70GDTNWVPPEMVER (SEQ ID NO: 608) 1564.76 SASGAYVLQWQNGGK (SEQ ID NO: 609)1735.86 GNFSLSGPLAGDTLTMR (SEQ ID NO: 610) 1750.86 EGVTNKDINSVFSWR(SEQ ID NO: 611) 1845.91 APNLYQTSEGYLLYSK (SEQ ID NO: 612) 1880.96ALGAYSLVGANVNYDINK (SEQ ID NO: 613) 1911.98 ALIEGIEASMAVPLMPDR(SEQ ID NO: 614) 2261.06 EIGLEFTVDDYHASVTYFR (SEQ ID NO: 615) 2416.14RPTNDWHGSLSLYTNQPESSK (SEQ ID NO: 616) 2701.36 SEISALYVEDNIEPMAGTNIIPGLR(SEQ ID NO: 617) 2909.36 FDYLSESGSNFSPSLNLSQELGEYVK (SEQ ID NO: 618)Dublin-3 81 605.33 NAVFR (SD3) (SEQ ID NO: 619) 616.37 VPVFR(SEQ ID NO: 620) 989.50 VPFNEAWK (SEQ ID NO: 621) 1063.48 YFMAVDYR(SEQ ID NO: 622) 1177.52 QDRDSDSLDK (SEQ ID NO: 623) 1314.62 LSLNYTYNDGR(SEQ ID NO: 624) 1329.77 IFEPLALTTGIR (SEQ ID NO: 625) 1526.73EVPGVQLTNEGDNR (SEQ ID NO: 626) 1649.90 GLDSSYTLILIDGKR (SEQ ID NO: 627)1740.90 DAPASISVITQQDLQR (SEQ ID NO: 628) 1744.69 MDDHETYGDHWSPR(SEQ ID NO: 629) 1750.84 WHGSVTVDSTIQEHR (SEQ ID NO: 630) 1792.88GEEGILEGVEASVTTFR (SEQ ID NO: 631) 1814.85 TSASQYALFLEDEWR(SEQ ID NO: 632) 1906.92 TPGGYVVWDTGAAWQATK (SEQ ID NO: 633) 1934.88EKDEQQSSATTATGETPR (SEQ ID NO: 634) 1952.94 HNDFDLNWIPVDAIER(SEQ ID NO: 635) 2196.14 YVLPLASVNQFLTFGGEWR (SEQ ID NO: 636) 2242.03TPDVNAAPGYSNFVGFETNSR (SEQ ID NO: 637) 2551.23 ADSATAKTPGGYVVWDTGAAWQATK(SEQ ID NO: 638) 2587.24 DRGDTYNGQFFTSGPLIDGVLGMK (SEQ ID NO: 639)2710.17 DGNVEFAWTPNENHDVTAGYGFDR (SEQ ID NO: 640) Dublin-4 61 631.37LSSGLR (SD4) (SEQ ID NO: 641) 944.50 SSLGAIQNR (SEQ ID NO: 642) 1100.52DDAAGQAIANR (SEQ ID NO: 643) 1115.56 SIQDEIQQR (SEQ ID NO: 644) 1163.58SQSSLSSAIER (SEQ ID NO: 645) 1316.67 SLGLDGFNVNGPK (SEQ ID NO: 646)1473.77 STANPLASIDSALSK (SEQ ID NO: 647) 1763.81 NVTGYDTYAAGADKYR(SEQ ID NO: 648) 1832.86 NVYTSVVNGQFTFDDK (SEQ ID NO: 649) 2007.00FDSAITNLGNTVTNLNSAR (SEQ ID NO: 650) 2084.12 AQVINTNSLSLLTQNNLNK(SEQ ID NO: 651) 2669.30 NANDGISIAQTTEGALNEINNNLQR (SEQ ID NO: 652)2682.28 VYVNAANGQLTTDDAENNTAVDLFK (SEQ ID NO: 653) 2859.59AQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 654) Dublin-5 56 913.53 NLSLLQAR(SD5) (SEQ ID NO: 655) 988.49 QLDQTTQR (SEQ ID NO: 656) 1285.63NNLDNAVEELR (SEQ ID NO: 657) 1381.76 YTYLINQLNIK (SEQ ID NO: 658)1549.77 AQYDTVLANEVTAR (SEQ ID NO: 659) 1615.87 FNVGLVAITDVQNAR(SEQ ID NO: 670) 1661.90 VLNAIDVLSYTQAQK (SEQ ID NO: 671) 1737.90TIVDVLDATTTLYDAK (SEQ ID NO: 672) 1828.90 SSFNNINASISSINAYK(SEQ ID NO: 673) 2033.96 QAQYNFVGASEQLESAHR (SEQ ID NO: 674) 2184.09SPLLPQLGLGADYTYSNGYR (SEQ ID NO: 675) 2208.09 QVTGNYYPELASLNVEHFK(SEQ ID NO: 676) Dublin-6 51 944.50 SSLGAIQNR (SD6) (SEQ ID NO: 677)1115.56 SIQDEIQQR (SEQ ID NO: 678) 1220.61 VSNQTQFNGVK (SEQ ID NO: 679)1316.67 SLGLDGFNVNGPK (SEQ ID NO: 680) 1444.65 NVTGYDTYAAGADK(SEQ ID NO: 681) 1473.77 STANPLASIDSALSK (SEQ ID NO: 682) 1813.94IQVGANDGETITIDLQK (SEQ ID NO: 683) 1832.86 NVYTSVVNGQFTFDDK(SEQ ID NO: 684) 2007.00 FDSAITNLGNTVTNLNSAR (SEQ ID NO: 685) 2669.30NANDGISIAQTTEGALNEINNNLQR (SEQ ID NO: 686) 2682.28VYVNAANGQLTTDDAENNTAVDLFK (SEQ ID NO: 687) 2859.59AQILQQAGTSVLAQANQVPQNVLSLL (SEQ ID NO: 688) Dublin-7 43 1171.64ELVNMIVAQR (SD7) (SEQ ID NO: 689) 1342.65 LVDSNGSVFYSR (SEQ ID NO: 691)1375.61 SGTASFADMFAGSK (SEQ ID NO: 692) 1422.73 GLDVAISQNGFFR(SEQ ID NO: 694) 1526.84 TQDQILNTLVNLR (SEQ ID NO: 695) 1853.88VAGITQDFTDGTTTNTGR (SEQ ID NO: 696) 2344.08 GTVTVYDSQGNAHDMNVYFVK(SEQ ID NO: 697) 3078.44 TKDNEWAVYTHDSSDPAATAPTTASTTLK (SEQ ID NO: 699)Dublin-8 40 1204.51 FADAGSFDYGR (SD8) (SEQ ID NO: 700) 1347.71INLLDKNDFTR (SEQ ID NO: 701) 1438.68 YVDVGATYYFNK (SEQ ID NO: 702)1800.82 DISNGYGASYGDQDIVK (SEQ ID NO: 703) 1834.81 FGTSNGSNPSTSYGFANK(SEQ ID NO: 704) 2247.08 NTDFFGLVDGLDFALQYQGK (SEQ ID NO: 705) 2339.08YDANNIYLAAQYSQTYNATR (SEQ ID NO: 706) 2405.02 VDGLHYFSDDKGSDGDQTYMR(SEQ ID NO: 707) 3004.51 AQNFEVVAQYQFDFGLRPSVAYLQSK (SEQ ID NO: 708)Dublin-10 38 817.43 LGGMVWR (SD9) (SEQ ID NO: 709) 1263.65 DGSVVVLGFTDR(SEQ ID NO: 710) 1639.81 LGYPITDDLDVYTR (SEQ ID NO: 711) 2302.20FGQQEAAPVVAPAPAPAPEVQTK (SEQ ID NO: 712) 2615.29DHDTGVSPVFAGGIEYAITPEIATR (SEQ ID NO: 713) 2672.37STLKPEGQQALDQLYSQLSNLDPK (SEQ ID NO: 714) 3422.69LEYQWTNNIGDANTIGTRPDNGLLSVGVSYR (SEQ ID NO: 715) ¹Molecular weight asdetermined by SDS-PAGE. ²The mass of a polypeptide fragment can beconverted to m/z value by adding 1 to the mass. Each mass includes arange of plus or minus 300 ppm for the polypeptide fragments from the 96kDa reference polypeptide; plus or minus Dalton for the polypeptidefragments from the 89 kDa, 81 kDa, 61 kDa, 56 kDa, 51 kDa, 40 kDa, and38 kDa reference polypeptides; and plus or minus 450 ppm for thepolypeptide fragments from the 43 kDa polypeptide.

TABLE 6 Characteristics of polypeptides obtained from an E. coli.mass value of predicted approximate polypeptide amino acid molecularfragments sequence of weight in resulting the polypeptide kilodaltonsfrom trypsin polypeptide designation (kDa)¹ digest² fragment Lw118 90628.39 IEVLR (SEQ ID NO: 716) 771.42 DINGVVR (SEQ ID NO: 717) 830.45DVSEIIR (SEQ ID NO: 718) 990.55 EIGLEFKR (SEQ ID NO: 719) 1177.61AGTYATTLPAGR (SEQ ID NO: 720) 1284.56 TWYMSVNTHF (SEQ ID NO: 721)1320.66 DGWLAGITWFR (SEQ ID NO: 722) 1367.75 NPVARDVSEIIR(SEQ ID NO: 723) 1432.77 YGNGAAGGVVNIITK (SEQ ID NO: 724) 1515.70GDTSWVPPEMIER (SEQ ID NO: 725) 1618.77 TMPGVNLTGNSTSGQR (SEQ ID NO: 726)1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 727) 1705.77 EDLSMQTTFTWYGK(SEQ ID NO: 728) 1786.85 TNFSLTGPLGDEFSFR (SEQ ID NO: 729) 1796.82TQADAWDINQGHQSAR (SEQ ID NO: 730) 1870.95 APSLYQTNPNYILYSK(SEQ ID NO: 731) 1965.01 EISPYSIVGLSATWDVTK (SEQ ID NO: 732) 1980.96WDFAPLQSLELEAGYSR (SEQ ID NO: 733) 2087.91 QGNLYAGDTQNTNSDAYTR(SEQ ID NO: 734) 2173.92 GSGEWHGSWDAYFNAPEHK (SEQ ID NO: 735) 2302.02KGSGEWHGSWDAYFNAPEHK (SEQ ID NO: 736) 2600.24 LYGNLDKTQADAWDINQGHQSAR(SEQ ID NO: 737) 2706.33 IEAGYVAVGQNAVGTDLYQWDNVPK (SEQ ID NO: 738)2843.31 AGNAQTTGDLAGANYIAGAGAYTYNEPGR (SEQ ID NO: 739) 3081.49FDHHSIVGNNWSPALNISQGLGDDFTLK (SEQ ID NO: 740) 3212.43QNYSLTWNGGWDNGVTTSNWVQYEHTR (SEQ ID NO: 741) Lw119 86 975.44 DPANSGPYR(SEQ ID NO: 742) 991.55 QVVATATFR (SEQ ID NO: 743) 1094.51 DDKQFTWR(SEQ ID NO: 744) 1246.63 RLPTDFNEGAK (SEQ ID NO: 745) 1277.70VGSYTVVDALVR (SEQ ID NO: 746) 1358.69 GASNTYDHLIIR (SEQ ID NO: 747)1395.61 YDWADQESLNR (SEQ ID NO: 748) 1435.73 EALSYTPGVSVGTR(SEQ ID NO: 749) 1492.65 YTGSSYGDPANSFK (SEQ ID NO: 750) 1571.87RPTTEPLKEVQFK (SEQ ID NO: 751) 1649.82 YAIAPAFTWRPDDK (SEQ ID NO: 752)1664.78 QTGVYVQDQAQWDK (SEQ ID NO: 753) 1809.86 GFAAEGQSQNNYLNGLK(SEQ ID NO: 754) 1912.96 VGMAGSNVALHVNNLFDR (SEQ ID NO: 755) 2133.15YVPEDRPIVVTGAVYNLTK (SEQ ID NO: 756) 2209.96 TSQNSVYGYGVCSDPANAYSK(SEQ ID NO: 757) 2269.03 TNNLMADPEGSFFSVEGGEIR (SEQ ID NO: 758) LW-1A-383 836.45 TLRYER (SEQ ID NO: 759) 883.47 WGLAGQPR (SEQ ID NO: 760)1047.46 SEQRDGDNK (SEQ ID NO: 761) 1126.59 DKWGLAGQPR (SEQ ID NO: 762)1337.62 DGQLGSLTGGYDR (SEQ ID NO: 763) 1350.68 DGVVLASTGETFR(SEQ ID NO: 764) 1396.63 SYLNWNETENK (SEQ ID NO: 765) 1471.73FTQNYSSLSAVQK (SEQ ID NO: 766) 1604.85 GMPASYTLILIDGVR (SEQ ID NO: 767)1649.85 AYLVWDVADAWTLK (SEQ ID NO: 768) 1721.84 IPYPTESQNYNLGAR(SEQ ID NO: 769) 1726.76 YEHHEQFGGHFSPR (SEQ ID NO: 770) 1758.89WLSSVNAGLNLQESNK (SEQ ID NO: 771) 1812.80 ASEQDVLWFDMDTTR(SEQ ID NO: 772) 2266.14 GPMSTLYGSDAMGGVVNIITRK (SEQ ID NO: 774) 2512.18STLYAGDYFQTGSSTTGYVIPER (SEQ ID NO: 775) Lw121 79 715.38 HFSIGR(SEQ ID NO: 776) 951.48 IYWSEVR (SEQ ID NO: 777) 1134.55 IWGWDVMTK(SEQ ID NO: 778) 1154.67 DLPITLLGGTR (SEQ ID NO: 779) 1221.63 MSRPQFTSLR(SEQ ID NO: 780) 1335.64 DLLQEGQSSGFR (SEQ ID NO: 781) 1395.64INAQNTGSSGEYR (SEQ ID NO: 782) 1446.71 TENLDGIVAWSSR (SEQ ID NO: 783)1511.74 LAPQGNDWLNADAK (SEQ ID NO: 784) 1531.71 EYWSPQGIPQDGR(SEQ ID NO: 785) 1652.79 QEQHPGGATTGFPQAK (SEQ ID NO: 786) 1656.77FDDLMLSNDALEFK (SEQ ID NO: 787) 1676.80 YTTDLFSLDVAYNR (SEQ ID NO: 788)1716.88 GTWQIDSAQSLSGLVR (SEQ ID NO: 789) 1778.88 IDFSSGWLQDEITLR(SEQ ID NO: 790) 1859.83 NPQTVEASESSNPMVDR (SEQ ID NO: 791) 1962.95VFGTGGTGDHSLGLGASAFGR (SEQ ID NO: 792) 2261.08 QPGYGVNDFYVSYQGQQALK(SEQ ID NO: 793) 2397.13 STLFADSFASHLLTYGGEYYR (SEQ ID NO: 794) LW-1A-5A66 631.37 LSSGLR (SEQ ID NO: 795) 944.50 SSLGAIQNR (SEQ ID NO: 796)1190.59 NQSALSSSIER (SEQ ID NO: 797) 1237.66 LNTTTGLYDLK(SEQ ID NO: 798) 1439.81 AQIIQQAGNSVLAK (SEQ ID NO: 799) 1560.83VSGQTQFNGVNVLAK (SEQ ID NO: 800) 1646.83 DYAPAIGTAVNVNSAGK(SEQ ID NO: 801) 1790.92 KIDSDTLGLNGFNVNGK (SEQ ID NO: 802) 2060.95TIGFTAGESSDAAKSYVDDK (SEQ ID NO: 803) 2084.12 AQVINTNSLSLITQNNINK(SEQ ID NO: 804) 2189.01 AASEGSDGASLTFNGTEYTIAK (SEQ ID NO: 805) 2248.09LDSAVTNLNNTTTNLSEAQSR (SEQ ID NO: 806) 2454.34 ATPATTTPVAPLIPGGITYQATVSK(SEQ ID NO: 807) 2628.27 NANDGISVAQTTEGALSEINNNLQR (SEQ ID NO: 808)LW-1A-5B 66 678.37 VEYIR (SEQ ID NO: 809) 1294.63 LYSQSWDAGLR(SEQ ID NO: 810) 1298.53 DYNYDPHYGR (SEQ ID NO: 811) 1303.65 WQSTSVNDVLR(SEQ ID NO: 812) 1422.73 KLYSQSWDAGLR (SEQ ID NO: 813) 1549.86GTNASHVLVLIDGVR (SEQ ID NO: 814) 1819.86 QWEGAFEGLTAGVNWR(SEQ ID NO: 815) 1892.00 SAVYGSDAIGGVVNIITTR (SEQ ID NO: 816) 1917.87TNYDAYYSPGSPLVDTR (SEQ ID NO: 817) 2158.14 LPGVDITQNGGSGQLSSIFIR(SEQ ID NO: 818) 2323.11 IANLFDKDYETVYGYQTAGR (SEQ ID NO: 819) 2357.17NTGIYLTGLQQVGDFTFEGAAR (SEQ ID NO: 820) 2698.25GVEATANFDTGPLTHTVSYDYVDAR (SEQ ID NO: 821) LW-1A-6 56 1284.64NNLDNAVEQLR (SEQ ID NO: 822) 1394.76 YNYLINQLNIK (SEQ ID NO: 823)1549.77 AQYDTVLANEVTAR (SEQ ID NO: 824) 1615.87 FNVGLVAITDVQNAR(SEQ ID NO: 825) 1828.90 SSFNNINASISSINAYK (SEQ ID NO: 826) 2033.96QAQYNFVGASEQLESAHR (SEQ ID NO: 827) 2123.10 VGLSFSLPIYQGGMVNSQVK(SEQ ID NO: 828) 2183.09 QITGNYYPELAALNVENFK (SEQ ID NO: 829) 2226.05QAVVSAQSSLDAMEAGYSVGTR (SEQ ID NO: 830) Lw123 38 704.42 VAFAGLK(SEQ ID NO: 831) 884.41 GNGFATYR (SEQ ID NO: 832) 930.49 VGSLGWANK(SEQ ID NO: 833) 938.47 AETYTGGLK (SEQ ID NO: 834) 1119.49 NMSTYVDYK(SEQ ID NO: 835) 1121.57 DGNKLDLYGK (SEQ ID NO: 836) 1123.50 NYDDEDILK(SEQ ID NO: 837 1170.50 SVDGDQTYMR (SEQ ID NO: 838) 1289.57 FQDVGSFDYGR(SEQ ID NO: 839) 1347.68 INLLDDNQFTR (SEQ ID NO: 840) 1378.59NGSVSGEGMTNNGR (SEQ ID NO: 841) 1378.59 NGSVSGEGMTNNGR (SEQ ID NO: 842)1438.68 YVDVGATYYFNK (SEQ ID NO: 843) 1663.74 TDDQNSPLYIGNGDR(SEQ ID NO: 844) 1819.84 RTDDQNSPLYIGNGDR (SEQ ID NO: 845) 1819.84RTDDQNSPLYIGNGDR (SEQ ID NO: 846) 2232.09 NTDFFGLVDGLNFAVQYQGK(SEQ ID NO: 847) 2353.10 YDANNIYLAAQYTQTYNATR (SEQ ID NO: 848) 2447.07VDGLHYFSDDKSVDGDQTYMR (SEQ ID NO: 849) 2584.20 TDDQNSPLYIGNGDRAETYTGGLK(SEQ ID NO: 850) 2791.30 QNGDGVGGSITYDYEGFGIGAAVSSSKR (SEQ ID NO: 851)2990.49 AQNFEAVAQYQFDFGLRPSLAYLQSK (SEQ ID NO: 852) 3104.47EALRQNGDGVGGSITYDYEGFGIGAAVSSSK (SEQ ID NO: 853) Lw124 37 817.43 LGGMVWR(SEQ ID NO: 854) 914.52 AQGVQLTAK (SEQ ID NO: 855) 1026.58 GIPADKISAR(SEQ ID NO: 856) 1054.47 DNTWYTGAK (SEQ ID NO: 857) 1082.54 SDVLFNFNK(SEQ ID NO: 858) 1154.63 GIKDVVTQPQA (SEQ ID NO: 859) 1221.66AQSVVDYLISK (SEQ ID NO: 860) 1279.64 DGSVVVLGYTDR (SEQ ID NO: 861)1377.76 RAQSVVDYLISK (SEQ ID NO: 862) 1408.66 IGSDAYNQGLSER(SEQ ID NO: 863) 1442.69 MPYKGSVENGAYK (SEQ ID NO: 864) 1564.76IGSDAYNQGLSERR (SEQ ID NO: 865) 1653.82 LGYPITDDLDIYTR (SEQ ID NO: 866)1708.89 HFTLKSDVLFNFNK (SEQ ID NO: 867) 2231.16 FGQGEAAPVVAPAPAPAPEVQTK(SEQ ID NO: 868) 2599.35 ATLKPEGQAALDQLYSQLSNLDPK (SEQ ID NO: 869)2600.29 NHDTGVSPVFAGGVEYAITPEIATR (SEQ ID NO: 870) 2670.29DGSVVVLGYTDRIGSDAYNQGLSER (SEQ ID NO: 871) 3477.67LEYQWTNNIGDAHTIGTRPDNGMLSLGVSYR (SEQ ID NO: 872) LW-1A-10 29 950.52ATNLLYTR (SEQ ID NO: 873) 1019.54 YAIVANDVR (SEQ ID NO: 874) 1484.73TALIDHLDTMAER (SEQ ID NO: 875) 1516.89 QVIQFIDLSLITK (SEQ ID NO: 877)1675.81 GANFIAVHEMLDGFR (SEQ ID NO: 878) ¹Molecular weight as determinedby SDS-PAGE. ²The mass of a polypeptide fragment can be converted to m/zvalue by adding 1 to the mass. Each mass includes a range of plus orminus 300 ppm (the 83 kDa and 29 kDa polypeptides), 450 ppm (the 66 kDaand 56 kDa polypeptides), or 1 Dalton (the remaining polypeptide).

TABLE 7 Characteristics of polypeptides  obtained from an E. coli.mass value of predicted approximate polypeptide amino acid molecularfragments sequence of weight in resulting the polypeptide kilodaltonsfrom trypsin polypeptide designation (kDa)¹ digest² fragment AB1-1 92904.51 LVQLNYR (SEQ ID NO: 880) 908.44 GGIQYDTR (SEQ ID NO: 881) 1050.50IYDDAAVER (SEQ ID NO: 882) 1078.54 EAPGQPEPVR (SEQ ID NO: 883) 1171.60AQYLYVPYR (SEQ ID NO: 884) 1276.56 YGSSTDGYATQK (SEQ ID NO: 885) 1343.65EEQVAEIWNAR (SEQ ID NO: 886) 1403.73 IYGQAVHFVNTR (SEQ ID NO: 887)1450.67 RGNIMWENEFR (SEQ ID NO: 888) 1479.76 YASPEYIQATLPK(SEQ ID NO: 889) 1510.72 TGSLVWAGDTYWR (SEQ ID NO: 890) 1546.66VSDPSYFNDFDNK (SEQ ID NO: 891) 1567.75 LDNVATSNSSIEYR (SEQ ID NO: 892)1624.79 GLSSNYGLGTQEMLR (SEQ ID NO: 893) 1668.72 DTNVWEGDYQMVGR(SEQ ID NO: 895) 1739.90 NGISQVGAVASWPIADR (SEQ ID NO: 897) 1822.89FNISVGQIYYFTESR (SEQ ID NO: 898) 1858.93 QHAVYDNAIGFNIELR(SEQ ID NO: 899) 2122.02 LLATHYQQTNLDWYNSR (SEQ ID NO: 900) 2140.01IYNYDSSLLQSDYSGLFR (SEQ ID NO: 901) AB1-2 80 628.39 IEVLR(SEQ ID NO: 902) 830.45 DVSEIIR (SEQ ID NO: 903) 1177.61 AGTYATTLPAGR(SEQ ID NO: 904) 1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 905) 1786.85TNFSLTGPLGDEFSFR (SEQ ID NO: 906) 1796.82 TQADAWDINQGHQSAR(SEQ ID NO: 907) 1870.95 APSLYQTNPNYILYSK (SEQ ID NO: 908) 1980.96WDFAPLQSLELEAGYSR (SEQ ID NO: 909) 2087.91 QGNLYAGDTQNTNSDAYTR(SEQ ID NO: 910) 2091.10 LSIIPEYTLNSTLSWQAR (SEQ ID NO: 911) 2173.92GSGEWHGSWDAYFNAPEHK (SEQ ID NO: 912) 2302.02 KGSGEWHGSWDAYFNAPEHK(SEQ ID NO: 913) 2843.31 AGNAQTTGDLAGANYIAGAGAYTYNEPGR (SEQ ID NO: 914)AB1-3 77 1380.59 SSEGANTYNEPGR (SEQ ID NO: 915) 1525.72 GDTNWVPPEQVER(SEQ ID NO: 916) 1688.88 ANFSLSGPLAGNALTTR (SEQ ID NO: 917) 1748.79TNNTRMNEGLSGGGEGR (SEQ ID NO: 918) 1831.90 APNLYQSSEGYLLYSK(SEQ ID NO: 919) 1889.03 IVAGDNVIGQTASGAYILK (SEQ ID NO: 920) 1929.04GPAAARYGSGAAGGVVNII TK (SEQ ID NO:921) 1968.03 GMGPENTLILIDGVPVTSR(SEQ ID NO: 922) 2030.93 QNYGITHNGIWDWGQSR (SEQ ID NO: 924) 2917.36QNYGITHNGIWDWGQSRFGVYYEK (SEQ ID NO: 925) 2959.54NHSQISALYIEDNIEPVPGTNIIPGLR (SEQ ID NO: 926) AB1-4 72 628.39 IEVIR(SEQ ID NO: 927) 807.40 YWANVR (SEQ ID NO: 928) 871.47 QIWAAQR(SEQ ID NO: 929) 888.44 FVFEYGK (SEQ ID NO: 930) 1738.77 DKYNHWDLNYESR(SEQ ID NO: 931) 1762.81 LDNHEIYGSYWNPR (SEQ ID NO: 932) 1872.93FGNSTTNDFYLSGPLIK (SEQ ID NO: 933) 1999.02 DVEGISITGGNEKPDISIR(SEQ ID NO: 934) 2103.95 SEDIDTIDGNWQVDEGRR (SEQ ID NO: 935) 2141.02EVSPGFGTLTQGGASIMYGNR (SEQ ID NO: 936) 2207.09 DPVTGLNTFIYDNVGEANIR(SEQ ID NO: 937) 2415.18 ESRPNGSGGFEAGFIPPVEAIER (SEQ ID NO: 938)2439.29 VTAFLPENVLTIGGQFQHAELR (SEQ ID NO: 939) AB1-5 66 614.38 VEIVR(SEQ ID NO: 940) 715.38 HFSIGR (SEQ ID NO: 941) 770.48 RVEIVR(SEQ ID NO: 942) 830.38 ASYFDTK (SEQ ID NO: 943) 941.46 IFVSYQW(SEQ ID NO: 944) 951.48 IYWSEVR (SEQ ID NO: 945) 1025.62 GVLVLVDGVR(SEQ ID NO: 946) 1134.55 IWGWDVMTK (SEQ ID NO: 947) 1154.67 DLPITLLGGTR(SEQ ID NO: 948) 1221.63 MSRPQFTSLR (SEQ ID NO: 949) 1335.64DLLQEGQSSGFR (SEQ ID NO: 950) 1395.64 INAQNTGSSGEYR (SEQ ID NO: 951)1531.71 EYWSPQGIPQDGR (SEQ ID NO: 952) 1656.77 FDDLMLSNDALEFK(SEQ ID NO: 953) 1676.80 YTTDLFSLDVAYNR (SEQ ID NO: 955) 1716.88GTWQIDSAQSLSGLVR (SEQ ID NO: 956) 1778.88 IDFSSGWLQDEITLR(SEQ ID NO: 957) 1962.95 VFGTGGTGDHSLGLGASAFGR (SEQ ID NO: 958) 1997.02QGTDTGHLNGTFLDPALIK (SEQ ID NO: 959) 2261.08 QPGYGVNDFYVSYQGQQALK(SEQ ID NO: 960) 2397.13 STLFADSFASHLLTYGGEYYR (SEQ ID NO: 961) 3304.56FYTNYWVPNPNLRPETNETQEYGFGLR (SEQ ID NO: 962) AB1-6 50 787.46 SVVQTVR(SEQ ID NO: 963) 801.43 LSQDLAR (SEQ ID NO: 964) 827.45 LSNPELR(SEQ ID NO: 965) 913.53 NLSLLQAR (SEQ ID NO: 966) 1179.55 SAADRDAAFEK(SEQ ID NO: 967) 1344.62 TTTSNGHNPFRN (SEQ ID NO: 968) 1736.92TIVDVLDATTTLYNAK (SEQ ID NO: 969) 1828.90 SSFNNINASISSINAYK(SEQ ID NO: 970) 2033.96 QAQYNFVGASEQLESAHR (SEQ ID NO: 971) 2033.96QAQYNFVGASEQLESAHR (SEQ ID NO: 972) 2183.09 QITGNYYPELAALNVENFK(SEQ ID NO: 973) 2183.09 QITGNYYPELAALNVENFK (SEQ ID NO: 974) 2184.09SPLLPQLGLGADYTYSNGYR (SEQ ID NO: 975) 2226.06 QAVVSAQSSLDAMEAGYSVGTR(SEQ ID NO: 976) 2226.06 QAVVSAQSSLDAMEAGYSVGTR (SEQ ID NO: 977) AB1-742 631.37 LSSGLR (SEQ ID NO: 978) 708.38 FTSNIK (SEQ ID NO: 979) 715.40GLTQAAR (SEQ ID NO: 980) 759.38 LDEIDR (SEQ ID NO: 981) 930.49 SSLGAVQNR(SEQ ID NO: 982) 1002.51 SRLDEIDR (SEQ ID NO: 983) 1019.54 AIAQVDTFR(SEQ ID NO: 984) 2248.09 LDSAVTNLNNTTTNLSEAQSR (SEQ ID NO: 985) 2642.29NANDGISLAQTTEGALSEINNNLQR (SEQ ID NO: 986) 2700.24GAATSQFVVQSGNDFYSASINHTDGK (SEQ ID NO: 987) 2814.50VVVELSTAKPTAQFSGASSADPLALLDK (SEQ ID NO: 988) AB1-8 38 704.42 VAFAGLK(SEQ ID NO: 989) 841.48 NLGVINGR (SEQ ID NO: 990) 884.41 GNGFATYR(SEQ ID NO: 991) 1289.57 FQDVGSFDYGR (SEQ ID NO: 992) 1438.68YVDVGATYYFNK (SEQ ID NO: 993) 2353.10 YDANNIYLAAQYTQTYNATR(SEQ ID NO: 994) 2990.49 AQNFEAVAQYQFDFGLRPSLAYLQSK (SEQ ID NO: 995)AB1-9 36 718.44 LAFAGLK (SEQ ID NO: 996) 867.42 DLGVHGDR(SEQ ID NO: 997) 1057.56 NAEVWAAGLK (SEQ ID NO: 998) 1248.54 FGDYGSIDYGR(SEQ ID NO: 999) 1438.68 YVDVGATYYFNK (SEQ ID NO: 1000) 1933.77HYFSSNDADDGDTTYAR (SEQ ID NO: 1001) 2217.05 NNDFFGLVDGLNFAAQYQGK(SEQ ID NO: 1002) 2389.09 GETQINDQLTGFGQWEYEFK (SEQ ID NO: 1003) 2834.36LGFKGETQINDQLTGFGQWEYEFK (SEQ ID NO: 1004) AB1-10 35 817.43 LGGMVWR(SEQ ID NO: 1005) 871.51 RVEIEVK (SEQ ID NO: 1007) 1054.47 DNTWYTGAK(SEQ ID NO: 1008) 1279.64 DGSVVVLGYTDR (SEQ ID NO: 1009) 1377.76RAQSVVDYLISK (SEQ ID NO: 1010) 1422.67 IGSDAYNQALSER (SEQ ID NO: 1011)1639.81 LGYPITDDLDVYTR (SEQ ID NO: 1012) 2231.16 FGQGEAAPVVAPAPAPAPEVQTK(SEQ ID NO: 1013) 2599.35 ATLKPEGQAALDQLYSQLSNLDPK (SEQ ID NO: 1014)3477.67 LEYQWTNNIGDAHTIGTRPDNGMLSLGVSYR (SEQ ID NO: 1015) AB1-11 30706.34 YWNPK (SEQ ID NO: 1016) 776.48 ILFVGTK (SEQ ID NO: 1017) 929.52LENSLGGIK (SEQ ID NO: 1018) 964.57 VHIINLEK (SEQ ID NO: 1019) 1065.58MKPFIFGAR (SEQ ID NO: 1020) 1108.55 AGVHFGHQTR (SEQ ID NO: 1022) 1204.61WLGGMLTNWK (SEQ ID NO: 1023) 1403.81 AVTLYLGAVAATVR (SEQ ID NO: 1025)1560.82 WLGGMLTNWKTVR (SEQ ID NO: 1026) 1575.79 TVPMFNEALAELNK(SEQ ID NO: 1027) 2376.17 DMGGLPDALFVIDADHEHIAIK (SEQ ID NO: 1028) Lw21419 914.52 ATVELLNR (SEQ ID NO: 1029) 941.43 QAHWNMR (SEQ ID NO: 1030)950.52 ATNLLYTR (SEQ ID NO: 1031) 1019.54 YAIVANDVR (SEQ ID NO: 1032)1484.73 TALIDHLDTMAER (SEQ ID NO: 1033) 1516.89 QVIQFIDLSLITK(SEQ ID NO: 1034) 1603.83 ELADRYAIVANDVR (SEQ ID NO: 1035) 1675.81GANFIAVHEMLDGFR (SEQ ID NO: 1036) 1677.85 SYPLDIHNVQDHLK(SEQ ID NO: 1037) 1677.85 SYPLDIHNVQDHLK (SEQ ID NO: 1038) 2413.39ATVELLNRQVIQFIDLSLITK (SEQ ID NO: 1039) Lw215 16 602.30 ELADR(SEQ ID NO: 1040) 914.52 ATVELLNR (SEQ ID NO: 1041) 941.43 QAHWNMR(SEQ ID NO: 1042) 950.52 ATNLLYTR (SEQ ID NO: 1043) 1019.54 YAIVANDVR(SEQ ID NO: 1044) 1042.61 KATVELLNR (SEQ ID NO: 1045) 1147.63 YAIVANDVRK(SEQ ID NO: 1046) 1362.63 DDDTADILTAASR (SEQ ID NO: 1047) 1484.73TALIDHLDTMAER (SEQ ID NO: 1048) 1516.89 QVIQFIDLSLITK (SEQ ID NO: 1049)1603.83 ELADRYAIVANDVR (SEQ ID NO: 1050) 1675.81 GANFIAVHEMLDGFR(SEQ ID NO: 1051) 1767.87 DLDKFLWFIESNIE (SEQ ID NO: 1052) 1931.94AIGEAKDDDTADILTAASR (SEQ ID NO: 1053) 2262.14 SYPLDIHNVQDHLKELADR(SEQ ID NO: 1054) ¹Molecular weight as determined by SDS-PAGE. ²The massof a polypeptide fragment can be converted to m/z value by adding 1 tothe mass. Each mass includes a range of plus or minus 250 ppm (the 92kDa polypeptide), plus or minus 300 ppm (the 80 kDa and 30 kDapolypeptides), plus or minus 400 ppm (the 77 kDa, 72 kDa, 42 kDa, and 35kDa polypeptides), plus or minus 450 ppm (the 50 kDa and 36 kDapolypeptides), plus or minus 500 ppm (the 66 kDa and 38 kDapolypeptides) or 1 Dalton (the 19 kDa and 16 kDa polypeptides).

TABLE 8 Characteristics of polypeptides obtained from an E. coli.mass value of predicted approximate polypeptide amino acid molecularfragments sequence of weight in resulting the polypeptide kilodaltonsfrom trypsin polypeptide designation (kDa)¹ digest² fragment J4-1 82628.39 IEVLR (SEQ ID NO: 1055) 1306.65 DGWLAGVTWFR (SEQ ID NO: 1056)1515.70 GDTSWVPPEMIER (SEQ ID NO: 1057) 1577.83 AETSINKEIGLEFK(SEQ ID NO: 1058) 1633.84 NVSLTGGVDNLFDKR (SEQ ID NO: 1059) 1786.85TNFSLTGPLGDEFSFR (SEQ ID NO: 1060) 1796.82 TQADAWDINQGHQSAR(SEQ ID NO: 1061) 1980.96 WDFAPLQSLELEAGYSR (SEQ ID NO: 1062) 2087.91QGNLYAGDTQNTNSDAYTR (SEQ ID NO: 1063) 2091.10 LSIIPEYTLNSTLSWQAR(SEQ ID NO: 1064) 2126.08 GDTSWVPPEMIERIEVLR (SEQ ID NO: 1065) 2843.31AGNAQTTGDLAGANYIAGAGAYTYNEPGR (SEQ ID NO: 1066) J4-2 79 685.41 ITLSPR(SEQ ID NO: 1067) 736.37 NYWVR (SEQ ID NO: 1068) 841.51 RITLSPR(SEQ ID NO: 1069) 860.45 GIYAGQPR (SEQ ID NO: 1070) 1146.60 IPGFMLWGAR(SEQ ID NO: 1071) 1207.57 WQSTRPYDR (SEQ ID NO: 1073) 1243.56ENDVFEHAGAR (SEQ ID NO: 1074) 1278.57 YLNESTHEMR (SEQ ID NO: 1075)1472.74 IDIGNWTITPGMR (SEQ ID NO: 1076) 1486.76 FNIQGFYTQTLR(SEQ ID NO: 1077) 1578.82 YGPQSVGGVVNFVTR (SEQ ID NO: 1078) 1615.78EDALTVVGDWLGDAR (SEQ ID NO: 1079) 1717.84 LASLGYQFQPDSQHK(SEQ ID NO: 1080) 2013.89 ADYDADRWQSTRPYDR (SEQ ID NO: 1081) 2035.91YYTATSSGQLPSGSSPYDR (SEQ ID NO: 1082) 2110.03 YSQIFMIGPSAHEVGVGYR(SEQ ID NO: 1083) J4-3 88 649.35 GFSAIR (SEQ ID NO: 1085) 649.35 GFSAIR(SEQ ID NO: 1086) 671.36 IPGTER (SEQ ID NO: 1087) 820.42 FTGNNLR(SEQ ID NO: 1088) 1123.53 VDSYELGWR (SEQ ID NO: 1089) 1278.78GRPLVVLVDGVR (SEQ ID NO: 1090) 1296.65 FYPFPTVNANK (SEQ ID NO: 1091)1324.64 IDDFIGYAQQR (SEQ ID NO: 1092) 1380.62 SQGDDDYGLNLGK(SEQ ID NO: 1093) 1423.72 IAGAVSGGNEHISGR (SEQ ID NO: 1094) 1550.73GTSTPFVSNGLNSDR (SEQ ID NO: 1095) 1702.85 ATAYIGWAPDPWSLR(SEQ ID NO: 1096) 1731.80 VQSTTSFDVSDAQGYK (SEQ ID NO: 1097) 2023.96APLYYSPGYGPASLYDYK (SEQ ID NO: 1098) 2019.88 QVTAFSSSQQDTDQYGMK(SEQ ID NO: 1099) 2234.98 GQPETMMEFEAGTKSGFSSSK (SEQ ID NO: 1100)2785.41 HLISLQYSDSAFLGQELVGQVYYR (SEQ ID NO: 1101) 2847.27FGGWFDGNGDATLLDNTQTGLQYSDR (SEQ ID NO: 1102) J4-4 60 675.33 FEQPR(SEQ ID NO: 1103) 678.37 VEYIR (SEQ ID NO: 1104) 1755.80 YDKDYSSYPYQTVK(SEQ ID NO: 1105) 1819.86 QWEGAFEGLTAGVNWR (SEQ ID NO: 1106) 1892.00SAVYGSDAIGGVVNIITTR (SEQ ID NO: 1107) 1930.95 QDIDRWQSTSVNDVLR(SEQ ID NO: 1108) 2023.92 HGTWQTSAGWEFIEGYR (SEQ ID NO: 1109) 2158.14LPGVDITQNGGSGQLSSIFIR (SEQ ID NO: 1110) 2206.07 APNLGQLYGFYGNPNLDPEK(SEQ ID NO: 1111) 2255.23 LNLAGVSGSADLSQFPIALVQR (SEQ ID NO: 1112)2323.11 IANLFDKDYETVYGYQTAGR (SEQ ID NO: 1113) 2357.17NTGIYLTGLQQVGDFTFEGAAR (SEQ ID NO: 1114) 2698.25GVEATANFDTGPLTHTVSYDYVDAR (SEQ ID NO: 1115) J4-5 54 787.46 SVVQTVR(SEQ ID NO: 1116) 801.43 LSQDLAR (SEQ ID NO: 1117) 827.45 LSNPELR(SEQ ID NO: 1118) 913.53 NLSLLQAR (SEQ ID NO: 1119) 1230.57 TTTSNGHNPFR(SEQ ID NO: 1120) 1284.64 NNLDNAVEQLR (SEQ ID NO: 1121) 1322.76TDKPQPVNALLK (SEQ ID NO: 1122) 1344.62 TTTSNGHNPFRN (SEQ ID NO: 1123)1549.77 AQYDTVLANEVTAR (SEQ ID NO: 1124) 1615.87 FNVGLVAITDVQNAR(SEQ ID NO: 1125) 1736.92 TIVDVLDATTTLYNAK (SEQ ID NO: 1126) 1828.90SSFNNINASISSINAYK (SEQ ID NO: 1127) 2033.96 QAQYNFVGASEQLESAHR(SEQ ID NO: 1128) 2123.10 VGLSFSLPIYQGGMVNSQVK (SEQ ID NO: 1129) 2183.09QITGNYYPELAALNVENFK (SEQ ID NO: 1130) 2226.05 QAVVSAQSSLDAMEAGYSVGTR(SEQ ID NO: 1131) J4-6 46 730.43 LSLAATR (SEQ ID NO: 1132) 858.46LGQEVWK (SEQ ID NO: 1133) 963.46 VDFHGYAR (SEQ ID NO: 1134) 1562.74FAYNINNNGHMLR (SEQ ID NO: 1135) 1660.77 FVVQYATDSMTSQGK(SEQ ID NO: 1137) 1683.90 NLIEWLPGSTIWAGK (SEQ ID NO: 1138) 1730.86DGWLFTAEHTQSVLK (SEQ ID NO: 1139) 2132.06 LAQMEINPGGTLELGVDYGR(SEQ ID NO: 1140) 2209.07 LGNECETYAELKLGQEVWK (SEQ ID NO: 1142) 2355.08WTPIMSTVMEIGYDNVESQR (SEQ ID NO: 1143) 3216.38SSEAGGSSSFASNNIYDYTNETANDVFDVR (SEQ ID NO: 1145) J4-7 45 785.41 YALTYR(SEQ ID NO: 1146) 1024.46 GNYSSDLNR (SEQ ID NO: 1147) 1029.51 SISIPDQDR(SEQ ID NO: 1148) 1254.61 ATSTSGDTLFQK (SEQ ID NO: 1149) 1496.68INEGPYQFESEGK (SEQ ID NO: 1150) 1504.74 FWLSAGTTYAFNK (SEQ ID NO: 1151)1586.77 TGIAFDDSPVPAQNR (SEQ ID NO: 1152) 1651.74 AYSGEGAIADDAGNVSR(SEQ ID NO: 1153) 1777.83 DASVDVGVSYMHGQSVK (SEQ ID NO: 1154) 1898.94LNNAWSFGLGFNAVYAR (SEQ ID NO: 1155) 1997.92 IALGTTYYYDDNWTFR(SEQ ID NO: 1156) Lw216 38 867.44 TTGVATYR (SEQ ID NO: 1157) 1248.54FGDYGSIDYGR (SEQ ID NO: 1158) 1438.68 YVDVGATYYFNK (SEQ ID NO: 1159)1933.77 HYFSSNDADDGDTTYAR (SEQ ID NO: 1160) 2217.05 NNDFFGLVDGLNFAAQYQGK(SEQ ID NO: 1161) 2389.09 GETQINDQLTGFGQWEYEFK (SEQ ID NO: 1162) 2602.22NNDFFGLVDGLNFAAQYQGKNDR (SEQ ID NO: 1163) 2976.48AQNFEAVAQYQFDFGLRPSVAYLQSK (SEQ ID NO: 1164) 3306.53YDANNIYLATTYSETQNMTVFGNNHIANK (SEQ ID NO: 1165) Lw217 37 817.43 LGGMVWR(SEQ ID NO: 1166) 1279.64 DGSVVVLGYTDR (SEQ ID NO: 1167) 1377.69ADTKANVPGGASYK (SEQ ID NO: 1168) 2231.16 FGQGEAAPVVAPAPAPAPEVQTK(SEQ ID NO: 1169) 2599.35 ATLKPEGQAALDQLYSQLSNLDPK (SEQ ID NO: 1170)2601.27 DHDTGVSPVFAGGVEYAITPEIATR (SEQ ID NO: 1171) J4-11 31 706.34YWNPK (SEQ ID NO: 1172) 776.48 ILFVGTK (SEQ ID NO: 1173) 929.52LENSLGGIK (SEQ ID NO: 1174) 964.57 VHIINLEK (SEQ ID NO: 1175) 1065.58MKPFIFGAR (SEQ ID NO: 1176) 1108.55 AGVHFGHQTR (SEQ ID NO: 1178) 1204.61WLGGMLTNWK (SEQ ID NO: 1179) 1403.81 AVTLYLGAVAATVR (SEQ ID NO: 1181)1575.80 TVPMFNEALAELNK (SEQ ID NO: 1182) 2376.17 DMGGLPDALFVIDADHEHIAIK(SEQ ID NO: 1184) J4-12 30 716.40 FGPQIR (SEQ ID NO: 1185) 1338.68LTNTDLSFGPFK (SEQ ID NO: 1186) 1462.66 NDTYLEYEAFAK (SEQ ID NO: 1187)1840.79 EWYFANNYIYDMGR (SEQ ID NO: 1188) 1881.92 GIWNHGSPLFMEIEPR(SEQ ID NO: 1190) 2262.94 YQWQNYGAANENEWDGYR (SEQ ID NO: 1192) 2824.20YWHDGGQWNDDAELNFGNGNFNVR (SEQ ID NO: 1193) 2867.41TNNSIASSHILALNYDHWHYSVVAR (SEQ ID NO: 1194) Lw218 19 914.52 ATVELLNR(SEQ ID NO: 1195) 941.43 QAHWNMR (SEQ ID NO: 1196) 950.52 ATNLLYTR(SEQ ID NO: 1197) 1019.54 YAIVANDVR (SEQ ID NO: 1198) 1362.63DDDTADILTAASR (SEQ ID NO: 1199) 1484.73 TALIDHLDTMAER (SEQ ID NO: 1200)1516.89 QVIQFIDLSLITK (SEQ ID NO: 1201) 1603.83 ELADRYAIVANDVR(SEQ ID NO: 1202) 1675.81 GANFIAVHEMLDGFR (SEQ ID NO: 1203) 1677.85SYPLDIHNVQDHLK (SEQ ID NO: 1204) 1754.99 AVQLGGVALGTTQVINSK(SEQ ID NO: 1205) 1931.94 AIGEAKDDDTADILTAASR (SEQ ID NO: 1206) 2262.14SYPLDIHNVQDHLKELADR (SEQ ID NO: 1207) Lw219 16 602.30 ELADR(SEQ ID NO: 1208) 914.52 ATVELLNR (SEQ ID NO: 1209) 941.43 QAHWNMR(SEQ ID NO: 1210) 950.52 ATNLLYTR (SEQ ID NO: 1211) 1019.54 YAIVANDVR(SEQ ID NO: 1212) 1042.61 KATVELLNR (SEQ ID NO: 1213) 1147.63 YAIVANDVRK(SEQ ID NO: 1214) 1362.63 DDDTADILTAASR (SEQ ID NO: 1215) 1484.73TALIDHLDTMAER (SEQ ID NO: 1216) 1516.89 QVIQFIDLSLITK (SEQ ID NO: 1217)1603.83 ELADRYAIVANDVR (SEQ ID NO: 1218) 1675.81 GANFIAVHEMLDGFR(SEQ ID NO: 1219) 1754.99 AVQLGGVALGTTQVINSK (SEQ ID NO: 1220) 1767.87DLDKFLWFIESNIE (SEQ ID NO: 1221) 1931.94 AIGEAKDDDTADILTAASR(SEQ ID NO: 1222) 2262.14 SYPLDIHNVQDHLKELADR (SEQ ID NO: 1223)¹Molecular weight as determined by SDS-PAGE. ²The mass of a polypeptidefragment can be converted to m/z value by adding 1 to the mass. Eachmass includes a range of plus or minus 300 ppm (the 88 kDa, 79 kDa, 60kDa, 38 kDa, and 31 kDa polypeptides), plus or minus 350 ppm (the 46 kDapolypeptide), plus or minus 400 ppm (the 82 kDa, 54 kDa, 45 kDa, and 30kDa polypeptides), or plus or minus 1 Dalton (the 37 kDa, 19 kDa and 16kDa polypeptides).

TABLE 9 Characteristics of polypeptides obtained from an E. coli.mass value of predicted approximate polypeptide amino acid molecularfragments sequence of weight in resulting the polypeptide kilodaltonsfrom trypsin polypeptide designation (kDa)¹ digest² fragment Lw189A 101888.41 YGYAYPR (SEQ ID NO: 1224) 986.54 LAGDLETLR (SEQ ID NO: 1225)998.45 GYFPTDGSR (SEQ ID NO: 1226) 1008.47 WGYGDGLGGK (SEQ ID NO: 1227)1113.56 DIHFEGLQR (SEQ ID NO: 1228) 1276.60 GLEDFYYSVGK(SEQ ID NO: 1229) 1338.66 ALFATGNFEDVR (SEQ ID NO: 1230) 1401.71VPGSPDQVDVVYK (SEQ ID NO: 1231) 1470.69 DEVPWWNVVGDR (SEQ ID NO: 1232)1519.80 ERPTIASITFSGNK (SEQ ID NO: 1233) 1527.72 LGFFETVDTDTQR(SEQ ID NO: 1234) 1698.85 GIYVTVNITEGDQYK (SEQ ID NO: 1235) 1758.82YDGDKAEQFQFNIGK (SEQ ID NO: 1236) 1770.75 TDDFTFNYGWTYNK(SEQ ID NO: 1237) 1953.86 EMPFYENFYAGGSSTVR (SEQ ID NO: 1238) 2145.04SYGTDVTLGFPINEYNSLR (SEQ ID NO: 1239) 2154.96 TDDFTFNYGWTYNKLDR(SEQ ID NO: 1240) 2238.15 LSGVEVSGNLAGHSAEIEQLTK (SEQ ID NO: 1241)2253.97 LFYNDFQADDADLSDYTNK (SEQ ID NO: 1242) 2911.42LGFFETVDTDTQRVPGSPDQVDVVYK (SEQ ID NO: 1243) Lw189B 101 904.51 LVQLNYR(SEQ ID NO: 1244) 1171.60 AQYLYVPYR (SEQ ID NO: 1245) 1276.56YGSSTDGYATQK (SEQ ID NO: 1246) 1294.58 GNIMWENEFR (SEQ ID NO: 1247)1307.68 LQADEVQLHQK (SEQ ID NO: 1248) 1343.65 EEQVAEIWNAR(SEQ ID NO: 1249) 1403.73 IYGQAVHFVNTR (SEQ ID NO: 1250) 1450.68RGNIMWENEFR (SEQ ID NO: 1251) 1546.66 VSDPSYFNDFDNK (SEQ ID NO: 1252)1668.72 DTNVWEGDYQMVGR (SEQ ID NO: 1253) 1717.73 VYEDEHPNDDSSRR(SEQ ID NO: 1254) 1763.82 WSIVGAYYYDTNANK (SEQ ID NO: 1255) 1822.89FNISVGQIYYFTESR (SEQ ID NO: 1256) 1831.91 FSVGYAVQNFNATVSTK(SEQ ID NO: 1257) 1858.93 QHAVYDNAIGFNIELR (SEQ ID NO: 1258) 2013.02TVDALGNVHYDDNQVILK (SEQ ID NO: 1259) 2088.13 VGPVPIFYSPYLQLPVGDK(SEQ ID NO: 1260) Lw190 88 1177.61 AGTYATTLPAGR (SEQ ID NO: 1261)1177.61 AGTYATTLPAGR (SEQ ID NO: 1262) 1306.65 DGWLAGVTWFR(SEQ ID NO: 1263) 1515.70 GDTSWVPPEMIER (SEQ ID NO: 1264) 1577.83AETSINKEIGLEFK (SEQ ID NO: 1265) 1633.84 NVSLTGGVDNLFDKR(SEQ ID NO: 1266) 1786.85 TNFSLTGPLGDEFSFR (SEQ ID NO: 1267) 1796.82TQADAWDINQGHQSAR (SEQ ID NO: 1268) 1870.95 APSLYQTNPNYILYSK(SEQ ID NO: 1269) 1980.96 WDFAPLQSLELEAGYSR (SEQ ID NO: 1270) 2126.08GDTSWVPPEMIERIEVLR (SEQ ID NO: 1271) 2173.92 GSGEWHGSWDAYFNAPEHK(SEQ ID NO: 1272) 2302.02 KGSGEWHGSWDAYFNAPEHK (SEQ ID NO: 1273) 2706.33IEAGYVAVGQNAVGTDLYQWDNVPK (SEQ ID NO: 1274) 2843.31AGNAQTTGDLAGANYIAGAGAYTYNEPGR (SEQ ID NO: 1275) 3081.49FDHHSIVGNNWSPALNISQGLGDDFTLK (SEQ ID NO: 1276) 3196.44QNYALTWNGGWDNGVTTSNWVQYEHTR (SEQ ID NO: 1277) Lw191 85 564.28 FWGR(SEQ ID NO: 1278) 685.41 ITLSPR (SEQ ID NO: 1279) 736.37 NYWVR(SEQ ID NO: 1280) 860.45 GIYAGQPR (SEQ ID NO: 1281) 861.43 TWELGTR(SEQ ID NO: 1282) 1146.60 IPGFMLWGAR (SEQ ID NO: 1283) 1207.57 WQSTRPYDR(SEQ ID NO: 1284) 1243.56 ENDVFEHAGAR (SEQ ID NO: 1285) 1278.57YLNESTHEMR (SEQ ID NO: 1286) 1278.57 YLNESTHEMR (SEQ ID NO: 1287)1329.64 NIFDQDYFIR (SEQ ID NO: 1288) 1486.76 FNIQGFYTQTLR(SEQ ID NO: 1289) 1578.82 YGPQSVGGVVNFVTR (SEQ ID NO: 1290) 1615.78EDALTVVGDWLGDAR (SEQ ID NO: 1291) 1650.83 EKGDTYGNLVPFSPK(SEQ ID NO: 1292) 1696.78 SYDDNNKGIYAGQPR (SEQ ID NO: 1293) 1717.84LASLGYQFQPDSQHK (SEQ ID NO: 1294) 2013.89 ADYDADRWQSTRPYDR(SEQ ID NO: 1295) 2035.91 YYTATSSGQLPSGSSPYDR (SEQ ID NO: 1296) 2110.03YSQIFMIGPSAHEVGVGYR (SEQ ID NO: 1297) 2221.11 ALNQYAAHSGFTLSVDASLTR(SEQ ID NO: 1298) 2408.09 YYTATSSGQLPSGSSPYDRDTR (SEQ ID NO: 172)2581.22 ETHNLMVGGTADNGFGTALLYSGTR (SEQ ID NO: 1299) 2683.31AIPQDFGIEAGVEGQLSPTSSQNNPK (SEQ ID NO: 1300) 2946.40SGTEAHAWYLDDKIDIGNWTITPGMR (SEQ ID NO: 1301) 3021.50YDLGTLTPTLDNVSIYASYAYVNAEIR (SEQ ID NO: 1302) 3144.41YAPDEVHTFNSLLQYYDGEADMPGGLSR (SEQ ID NO: 1303) Lw193 77 523.26 YDVK(SEQ ID NO: 1304) 649.35 GFSAIR (SEQ ID NO: 1305) 820.42 FTGNNLR(SEQ ID NO: 1306) 1123.53 VDSYELGWR (SEQ ID NO: 1307) 1159.59 TFGLNYSVLF(SEQ ID NO: 1308) 1278.78 GRPLVVLVDGVR (SEQ ID NO: 1309) 1296.65FYPFPTVNANK (SEQ ID NO: 1310) 1324.64 IDDFIGYAQQR (SEQ ID NO: 1311)1372.71 GRTFGLNYSVLF (SEQ ID NO: 1312) 1380.62 SQGDDDYGLNLGK(SEQ ID NO: 1313) 1423.72 IAGAVSGGNEHISGR (SEQ ID NO: 1314) 1509.83GIYGAAVNGHLPLTK (SEQ ID NO: 1315) 1550.73 GTSTPFVSNGLNSDR(SEQ ID NO: 1316) 1553.84 DALAQLIPGLDVSSR (SEQ ID NO: 1317) 1649.84LEGVKVDSYELGWR (SEQ ID NO: 1318) 1702.85 ATAYIGWAPDPWSLR(SEQ ID NO: 1319) 1924.06 ELKDALAQLIPGLDVSSR (SEQ ID NO: 1320) 2013.00QQAWLNFSQGVELPDPGK (SEQ ID NO: 1321) 2023.96 APLYYSPGYGPASLYDYK(SEQ ID NO: 1322) 2204.08 GTSTPFVSNGLNSDRIPGTER (SEQ ID NO: 1323)2251.05 YQYTENKIDDFIGYAQQR (SEQ ID NO: 1324) 2552.24QQAWLNFSQGVELPDPGKYYGR (SEQ ID NO: 1325) 2785.41HLISLQYSDSAFLGQELVGQVYYR (SEQ ID NO: 1326) 2847.27FGGWFDGNGDATLLDNTQTGLQYSDR (SEQ ID NO: 1327) 3194.53ATSADAIPGGSVDYDNFLFNAGLLMHITER (SEQ ID NO: 1328) 3335.65YPSYDITNLAAFLQSGYDINNLFTLNGGVR (SEQ ID NO: 1329) 3385.69HLISLQYSDSAFLGQELVGQVYYRDESLR (SEQ ID NO: 1330) Lw194 67 678.37 VEYIR(SEQ ID NO: 1331) 1112.62 NAITDTPLLR (SEQ ID NO: 1332) 1135.55FIASYGTSYK (SEQ ID NO: 1333) 1243.71 STVLAPTTVVTR (SEQ ID NO: 1334)1249.63 SQLITSYSHSK (SEQ ID NO: 1335) 1294.63 LYSQSWDAGLR(SEQ ID NO: 1336) 1298.53 DYNYDPHYGR (SEQ ID NO: 1337) 1303.65WQSTSVNDVLR (SEQ ID NO: 1338) 1349.61 DYSSYPYQTVK (SEQ ID NO: 1339)1422.73 KLYSQSWDAGLR (SEQ ID NO: 1340) 1521.67 DYETVYGYQTAGR(SEQ ID NO: 1341) 1549.86 GTNASHVLVLIDGVR (SEQ ID NO: 1342) 1755.80YDKDYSSYPYQTVK (SEQ ID NO: 1343) 1761.81 NDVSDLIDYDDHTLK(SEQ ID NO: 1344) 1886.83 QTTTPGTGYVEDGYDQR (SEQ ID NO: 1345) 1892.00SAVYGSDAIGGVVNIITTR (SEQ ID NO: 1346) 1931.89 TNYDAYYSPGSPLLDTR(SEQ ID NO: 1347) 2023.92 HGTWQTSAGWEFIEGYR (SEQ ID NO: 1348) 2158.14LPGVDITQNGGSGQLSSIFIR (SEQ ID NO: 1349) 2206.07 APNLGQLYGFYGNPNLDPEK(SEQ ID NO: 1350) 2255.23 LNLAGVSGSADLSQFPIALVQR (SEQ ID NO: 1351)2323.11 IANLFDKDYETVYGYQTAGR (SEQ ID NO: 12) Lw195 38 1057.56 NAEVWAAGLK(SEQ ID NO: 20) 1248.54 FGDYGSIDYGR (SEQ ID NO: 584) 1438.68YVDVGATYYFNK (SEQ ID NO: 591) 1933.77 HYFSSNDADDGDTTYAR (SEQ ID NO: 595)1960.03 TDTQVNAGKVLPEVFASGK (SEQ ID NO: 690) 2217.05NNDFFGLVDGLNFAAQYQGK (SEQ ID NO: 693) 2389.09 GETQINDQLTGFGQWEYEFK(SEQ ID NO: 698) 2976.48 AQNFEAVAQYQFDFGLRPSVAYLQSK (SEQ ID NO: 773)3340.53 YDANNIYLATTYSETQNMTVFADHFVANK (SEQ ID NO: 876) 3549.48SDFDNYTEGNGDGFGFSATYEYEGFGIGATYAK (SEQ ID NO: 879) Lw196 35 644.36 HFTLK(SEQ ID NO: 894) 817.43 LGGMVWR (SEQ ID NO: 896) 871.51 RVEIEVK(SEQ ID NO: 923) 914.52 AQGVQLTAK (SEQ ID NO: 954) 1054.47 DNTWYTGAK(SEQ ID NO: 1006) 1082.54 SDVLFNFNK (SEQ ID NO: 1021) 1154.63GIKDVVTQPQA (SEQ ID NO: 1024) 1213.61 AALIDCLAPDR (SEQ ID NO: 1072)1221.66 AQSVVDYLISK (SEQ ID NO: 1084) 1232.63 LGGMVWRADTK(SEQ ID NO: 1136) 1279.64 DGSVVVLGYTDR (SEQ ID NO: 1141) 1369.71AALIDCLAPDRR (SEQ ID NO: 1144) 1377.76 RAQSVVDYLISK (SEQ ID NO: 1177)1408.66 IGSDAYNQGLSER (SEQ ID NO: 1180) 1653.82 LGYPITDDLDIYTR(SEQ ID NO: 1183) 2062.92 GMGESNPVTGNTCDNVKQR (SEQ ID NO: 1189) 2231.16FGQGEAAPVVAPAPAPAPEVQTK (SEQ ID NO: 1191) 2600.29NHDTGVSPVFAGGVEYAITPEIATR (SEQ ID NO: 173) 3477.67LEYQWTNNIGDAHTIGTRPDNGMLSLGVSYR (SEQ ID NO: 179) ¹Molecular weight asdetermined by SDS-PAGE. ²The mass of a polypeptide fragment can beconverted to m/z value by adding 1 to the mass. Each mass includes arange of plus or minus 150 ppm (the 38 kDa and 35 kDa polypeptide), plusor minus 300 ppm (the 101 kDa polypeptides), or plus or minus 1 Dalton(the 88 kDa, 85 kDa, 77 kDa, and 67 kDa polypeptides).

In yet another aspect, the present invention further includespolypeptides having similarity with an amino acid sequence. Thesimilarity is referred to as structural similarity and is generallydetermined by aligning the residues of the two amino acid sequences(i.e., a candidate amino acid sequence and a reference amino acidsequence) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. Reference amino acid sequencesare disclosed in Tables 10, 11, 12, 13, 14, 15, 16, and 17. Two aminoacid sequences can be prepared using commercially available algorithms.Preferably, two amino acid sequences are compared using the Blastpprogram of the BLAST 2 search algorithm, as described by Tatusova, etal., (FEMS Microbiol Lett 1999, 174:247-250). Preferably, the defaultvalues for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, acandidate amino acid sequence has at least 95% identity, at least 96%identity, at least 97% identity, at least 98% identity, or at least 99%identity to a reference amino acid sequence. Preferably, the molecularweight of the candidate amino acid sequence and the reference amino acidsequence are substantially the same value. Preferably, the molecularweight of the candidate amino acid sequence and the reference amino acidsequence is determined by SDS polyacrylamide gel electrophoresis. Acandidate polypeptide can be obtained by growth of a microbe under lowmetal conditions and the subsequent isolation of a polypeptide by theprocedures disclosed herein.

Typically, a candidate amino acid sequence having structural similarityto a reference amino acid sequence has seroreactive activity. As usedherein, “seroreactive activity” refers to the ability of a candidatepolypeptide to react with antibody present in convalescent serum from ananimal infected with an S. enterica serovar Newport (preferably,MS020508), an S. enterica serovar Enteritidis (preferably, MS010531), anS. enterica serovar Typhimurium (preferably, MS010427), an S. entericaserovar Dublin (preferably, IRP SDC Serial), or an E. coli (preferably,BEcO157(stx-), MS040330, MS040324, or MS040827). Preferably, when thecandidate polypeptide is compared to a reference polypeptide from table10, 11, 12, 13, 14, 15, 16, or 17, the convalescent serum is from ananimal infected with MS020508, MS010531, MS010427, IRP SDC Serial,BEc0157(stx-), MS040330, MS040324, or MS040827, respectively.

TABLE 10 S. enterica serovar Newport NCBI sequence identifier ofMolecular polypeptide identified by the weight of computer algorithm asreference having best match to mass polypeptide fingerprint of reference(kDa) polypeptide FIG. SEQ ID NO: 82 25300748 12 1367 80 20196197 131368 74 25300749 14 1369 65 16767394 15 1370 56 1706868 16 1371 5516226009 17 1372 52 25298420 18 1373 45 1941972 19 1374 38 16420094 201375 38 7428872 21 1376 36 25298549 22 1377 22 7162107 23 1378 1825301837 24 1379 12 25298585 25 1380

TABLE 11 S. enterica serovar Enteritidis Molecular NCBI sequenceidentifier of weight of polypeptide identified by the reference computeralgorithm as having polypeptide best match to mass fingerprint (kDa) ofreference polypeptide FIG. SEQ ID NO: 92 29136388 93 1381 91 25008880 261382 86 16419095 27 1383 83 20196197 28 1384 78 29136792 29 1385 5529139082 30 1386 40 19743622 31 1387 39 17865737 32 1388 38 20141670 331389

TABLE 12 S. enterica serovar Typhimurium Molecular NCBI sequenceidentifier of weight of polypeptide identified by the reference computeralgorithm as having polypeptide best match to mass fingerprint (kDa) ofreference polypeptide FIG. SEQ ID NO: 86 29138313 34 1390 82 16421325 351391 77 29136792 36 1392 40 16761195 37 1393 39 17865737 38 1394 3820141670 39 1395

TABLE 13 S. enterica serovar Dublin Molecular NCBI sequence identifierof weight of polypeptide identified by the reference computer algorithmas having polypeptide best match to mass fingerprint (kDa) of referencepolypeptide FIG. SEQ ID NO: 96 16419095 40 1396 89 16421325 41 1397 8116765529 42 1398 61 479267 43 1399 56 2495191 44 1400 51 531889 45 140143 16764533 46 1402 40 47797 47 1403 38 16764429 48 1404

TABLE 14 E. coli Molecular NCBI sequence identifier of weight ofpolypeptide identified by the reference computer algorithm as havingpolypeptide best match to mass fingerprint (kDa) of referencepolypeptide FIG. SEQ ID NO: 90 25300745 49 1405 86 15799834 50 1406 83 3661500 51 1407 79  1655877 52 1408 66 12516024 53 1409 66 15804564 541410 56 15803582 55 1411 38 25298428 56 1412 37 25298543 57 1413 29 232021 58 1414

TABLE 15 E. coli Molecular NCBI sequence identifier of weight ofpolypeptide identified by the reference computer algorithm as havingpolypeptide best match to mass fingerprint (kDa) of referencepolypeptide FIG. SEQ ID NO: 92 26106377 59 1415 80 26106960 60 1416 7725987939 61 1417 72 26250982 62 1418 66 13363854 63 1419 50 15803582 641420 42 6009835 65 1421 38 6650193 66 1422 36 26107830 67 1423 357188818 68 1424 30 26246115 69 1425 19 3660175 70 1426 16 232021 71 1427

TABLE 16 E. coli Molecular NCBI sequence identifier of weight ofpolypeptide identified by the reference computer algorithm as havingpolypeptide best match to mass fingerprint (kDa) of referencepolypeptide FIG. SEQ ID NO: 82 26106960 72 1428 79 7429053 73 1429 8878355 74 1430 60 7429052 75 1431 54 15803582 76 1432 46 3114532 77 143345 1799743 78 1434 38 26107830 79 1435 37 37624562 80 1436 31 1552746 811437 30 25348404 82 1438 19 3660175 83 1439 16 232021 84 1440

TABLE 17 E. coli Molecular NCBI sequence identifier of weight ofpolypeptide identified by the reference computer algorithm as havingpolypeptide best match to mass fingerprint (kDa) of referencepolypeptide FIG. SEQ ID NO: 101 21307716 85 1441 101 7430186 86 1442 886730010 87 1443 85 7429053 88 1444 77 38016693 89 1445 67 7429052 901446 38 33112659 91 1447 35 72585 92 1448

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably inactivated such that the cells cannot replicate, but theimmunogenic activity of the polypeptides of the present inventionexpressed by the microbe is maintained. Typically, the cells are killedby exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

Compositions

A composition of the present invention may include at least onepolypeptide described herein, or a number of polypeptides that is aninteger greater than 1 (e.g., at least 2, at least 3, at least 4, etc.)up to 15. A composition can include polypeptides obtainable from 1microbe, or can be obtainable from a combination of 2 or more microbes.For instance, a composition can include polypeptides obtainable from 2or more E. coli strains, or from 1 or more E. coli and 1 or moreSalmonella spp.

Optionally, a polypeptide of the present invention can be covalentlybound to a carrier polypeptide to improve the immunological propertiesof the polypeptide. Useful carrier polypeptides are known to the art.The chemical coupling of a polypeptide of the present invention can becarried out using known and routine methods. For instance, varioushomobifunctional and/or heterobifunctional cross-linker reagents such asbis(sulfosuccinimidyl) suberate, bis(diazobenzidine), dimethyladipimidate, dimethyl pimelimidate, dimethyl superimidate,disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl) cycloheane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (Harlow and Lane, Antibodies, A LaboratoryManual, generally and Chapter 5, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., N.Y. (1988)).

Preferably, such compositions of the present invention include lowconcentrations of lipopolysaccharide (LPS). LPS is a component of theouter membrane of most gram negative microbes (see, for instance,Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonellatyphimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.)American Society for Microbiology, Washington, D.C., pp. 7-22 (1987),and typically includes polysaccharides (O-specific chain, the outer andinner core) and the lipid A region. The lipid A component of LPS is themost biologically active component of the LPS structure and togetherinduce a wide spectrum of pathophysiological effects in mammals. Themost dramatic effects are fever, disseminated intravascular coagulation,complement activation, hypotensive shock, and death. The non-specificimmunostimulatory activity of LPS can enhance the formation of agranuloma at the site of administration of compositions that includeLPS. Such reactions can result in undue stress on the animal by whichthe animal may back off feed or water for a period of time, andexasperate infectious conditions in the animal. In addition, theformation of a granuloma at the site of injection can increase thelikelihood of possible down grading of the carcass due to scaring orblemishes of the tissue at the injection site (see, for instance, Rae,Injection Site Reactions, available atwww.animal.ufl.edu/short94/rae.htm).

The concentration of LPS can be determined using routine methods knownto the art. Such methods typically include measurement of dye binding byLPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189(1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, forinstance, Endotoxins and Their Detection With the Limulus AmebocyteLystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.(1982)). There are four basic commercially available methods that aretypically used with an LAL test: the gel-clot test; the turbidimetric(spectrophotometric) test; the colorimetric test; and the chromogenictest. An example of a gel-clot assay is available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma TechnicalBulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., EastFalmouth, Mass.). Typically, assay conditions include contacting thecomposition with a preparation containing a lysate of the circulatingamebocytes of the horseshoe crab, Limulus polyphemus. When exposed toLPS, the lysate increases in opacity as well as viscosity and may gel.About 0.1 milliliter of the composition is added to lysate. Typically,the pH of the composition is between 6 and 8, preferably, between 6.8and 7.5. The mixture of composition and lysate is incubated for about 1hour undisturbed at about 37° C. After incubation, the mixture isobserved to determine if there was gelation of the mixture. Gelationindicates the presence of endotoxin. To determine the amount ofendotoxin present in the composition, dilutions of a standardizedsolution of endotoxin are made and tested at the same time that thecomposition is tested. Standardized solutions of endotoxin arecommercially available from, for instance, Sigma Chemical (Catalog No.210-SE), U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503), andAssociates of Cape Cod, Inc., (Catalog No. E0005). In general, when acomposition of the present invention is prepared by isolatingpolypeptides from a microbe by a method as described herein (e.g., amethod that includes disrupting and solubilizing the cells, andcollecting the insoluble polypeptides), the amount of LPS in acomposition of the present invention is less than the amount of LPSpresent in a mixture of the same amount of the microbe that has beendisrupted under the same conditions but not solubilized. Typically, thelevel of LPS in a composition of the present invention is decreased by,in increasing order of preference, at least 50%, at least 60%, at least70%, at least 80%, or at least 90% relative to the level of LPS in acomposition prepared by disrupting, but not solubilizing, the samemicrobe.

The present invention also provides compositions including a whole cellpreparation of at least 1 Salmonella spp., at least about 1 E. coli, orthe combination thereof. In some aspects, a composition can includewhole preparations from 2, 3, 4, 5, or 6 E. coli strains.

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parental including intradermal, subcutaneous,intramuscular, intravenous, intraperitoneal, etc., and topically, suchas, intranasal, intrapulmonary, intramammary, intravaginal,intrauterine, intradermal, and rectally etc. It is foreseen that acomposition can be administered to a mucosal surface, such as byadministration to the nasal or respiratory mucosa (e.g. spray oraerosol), to stimulate mucosal immunity, such as production of secretoryIgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al.,(WO 96/01620 (1996)). Implants can be produced at sizes small enough tobe administered by aerosol or spray. Implants also include nanospheresand microspheres.

A composition of the present invention is administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of 10⁶ bacteria/ml, 10⁷ bacteria/ml, 10⁸bacteria/ml, or 10⁹ bacteria/ml. For an injectable composition (e.g.subcutaneous, intramuscular, etc.) the polypeptides may be present inthe composition in an amount such that the total volume of thecomposition administered is 0.5 ml to 5.0 ml, typically 1.0-2.0 ml. Whenthe composition is a whole cell preparation, the cells are preferablypresent in the composition in an amount that the total volume of thecomposition administered is 0.5 ml to 5.0 ml, typically 1.0-2.0 ml. Theamount administered will vary depending on various factors including,but not limited to, the specific polypeptides chosen, the weight,physical condition and age of the animal, and the route ofadministration. Thus, the absolute weight of the polypeptide included ina given unit dosage form can vary widely, and depends upon factors suchas the species, age, weight and physical condition of the animal, aswell as the method of administration. Such factors can be determined byone of skill in the art. Other examples of dosages suitable for theinvention are disclosed in Emery et al., (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., a polypeptide or whole cell of the present invention) intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.), ISA-70, RIBI and other substances known in the art.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known to the art such asan antibiotic, a preservative, an anti-oxidant, or a chelating agent.

Methods of Making

The polypeptides and whole cells of the present invention are obtainablefrom a member of the family Enterobacteriaceae, for instance, a memberof the tribe Escherichieae or Salmonelleae. Preferred examples ofmembers of the tribe Escherichieae are E. coli and Salmonella spp. ASalmonella spp. can be a member of serogroup A, B, C₁, C₂, C₃, D₁, D₂,D₃, E₁, E₂, E₃, E₄, G₁, G₂, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V,W, X, Y, Z, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. Preferredexamples of Salmonella spp. are S. cholerasuis, S. typhi, or one of theS. enterica serovars, e.g., Bredeney, Dublin, Agona, Blockley,Enteriditis, Typhimurium, Hadar, Heidelberg, Montevideo, Muenster,Newport, or Senftenberg, most preferably, S. enterica serovar Newport,S. enterica serovar Enteritidis, S. enterica serovar Typhimurium, and S.enterica serovar Dublin. Microbes useful for obtaining polypeptides ofthe present invention and making whole cell preparations arecommercially available from a depository such as American Type CultureCollection (ATCC). In addition, such microbes are readily obtainable bytechniques routine and known to the art. The microbes may be derivedfrom an infected animal as a field isolate, and used to obtainpolypeptides and/or whole cell prearations of the present invention, orstored for future use, for example, in a frozen repository at −20° C. to−95° C., or −40° C. to −50° C., in bacteriological media containing 20%glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from amicrobe, the microbe can be incubated under low metal conditions. Asused herein, the phrase “low metal conditions” refers to an environment,typically bacteriological media, that contains amounts of a free metalthat cause a microbe to express metal regulated polypeptides. As usedherein, the phrase “high metal conditions” refers to an environment thatcontains amounts of a free metal that cause a microbe to either notexpress one or more of the metal regulated polypeptides describedherein, or to decrease expression of such a polypeptide. Metals arethose present in the periodic table under Groups 1 through 17 (IUPACnotation; also referred to as Groups I-A, II-A, IV-B, V-B, VI-B, VII-B,VIII, I-B, II-B, III-A, IV-A, V-A, VI-A, and VII-A, respectively, underCAS notation). Preferably, metals are those in Groups 2 through 12, morepreferably, Groups 3-12. Even more preferably, the metal is iron, zinc,copper, magnesium, nickel, cobalt, manganese, molybdenum, or selenium,most preferably, iron.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium. High metal conditionsare generally present when a chelator is not present in the medium, ametal is added to the medium, or the combination thereof. Examples ofmetal chelators include natural and synthetic compounds. Examples ofnatural compounds include plant phenolic compounds, such as flavenoids.Examples of flavenoids include the copper chelators catechin andnaringenin, and the iron chelators myricetin and quercetin. Examples ofsynthetic copper chelators include, for instance, tetrathiomolybdate,and examples of synthetic zinc chelators include, for instance,N,N,N′,N′-Tetrakis (2-pyridylmethyl)-ethylene diamine. Examples ofsynthetic iron chelators include 2,2′-dipyridyl (also referred to in theart as α,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as, the catecholates and hydroxamates, andcitrate. Preferably, 2,2′-dipyridyl is used for the chelation of iron.Typically, 2,2′-dipyridyl is added to the media at a concentration of atleast 0.0025 micrograms/milliliter (μg/ml), at least 0.025 μg/ml, or atleast 0.25 μg/ml. High levels of 2,2′-dipyridyl can be 10 μg/ml, 20μg/ml, or 30 μg/ml.

It is expected that a Salmonella spp. or E. coli with a mutation in afur gene will result in the constitutive expression of many, if not all,of the metal regulated polypeptides of the present invention. Theproduction of a fur mutation in a Salmonella spp. or E. coli can beproduced using routine methods including, for instance, transposon,chemical, or site-directed mutagenesis useful for generating geneknock-out mutations in gram negative bacteria.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a microbe is being evaluated for theability to produce one or more of the polypeptides described herein, themicrobe can be grown in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine and known to the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferablybetween 6.9 and 7.1, and a temperature of 37° C.

In some aspects of the invention, a microbe may be harvested aftergrowth. Harvesting includes concentrating the microbe into a smallervolume and suspending in a media different than the growth media.Methods for concentrating a microbe are routine and known to the art,and include, for example, filtration or centrifugation. Typically, theconcentrated microbe is suspended in decreasing amounts of buffer.Preferably, the final buffer includes a metal chelator, preferably,ethylenediaminetetraacetic acid (EDTA). An example of a buffer that canbe used contains Tris-base (7.3 grams/liter) and EDTA (0.9 grams/liter),at a pH of 8.5. Optionally, the final buffer also minimizes proteolyticdegradation. This can be accomplished by having the final buffer at a pHof greater than 8.0, preferably, at least 8.5, and/or including one ormore proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride).Optionally and preferably, the concentrated microbe is frozen at −20° C.or below until disrupted.

When the microbe is to be used as a whole cell preparation, theharvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a microbe is to be used toprepare polypeptides of the present invention, the microbe may bedisrupted using chemical, physical, or mechanical methods routine andknown to the art, including, for example, french press, sonication, orhomoginization. Preferably, homoginization is used. As used herein,“disruption” refers to the breaking up of the cell. Disruption of amicrobe can be measured by methods that are routine and known to theart, including, for instance, changes in optical density. Typically, amicrobe is subjected to disruption until the percent transmittance isincreased by 20% when a 1:100 dilution is measured. The temperatureduring disruption is typically kept low, preferably at 4° C., to furtherminimize proteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, ananionic, zwitterionic, nonionic, or cationic detergent. Preferably, thedetergent is sarcosine, more preferably, sodium lauroyl sarcosinate. Asused herein, the term “solubilize” refers to dissolving cellularmaterials (e.g., polypeptides, nucleic acids, carbohydrates) into theaqueous phase of the buffer in which the microbe was disrupted, and theformation of aggregates of insoluble cellular materials. The conditionsfor solubilization preferably result in the aggregation of polypeptidesof the present invention into insoluble aggregates that are large enoughto allow easy isolation by, for instance, centrifugation.

Preferably, the sarcosine is added such that the final ratio ofsarcosine to gram weight of disrupted microbe is between 1.0 gramsarcosine per 4.5 grams pellet mass and 6.0 grams sarcosine per 4.5grams pellet mass, preferably, 4.5 gram sarcosine per 4.5 grams pelletmass. The solubilization of the microbe may be measured by methods thatare routine and known to the art, including, for instance, changes inoptical density. Typically, the solubilization is allowed to occur forat least 24 hours, more preferably, at least 48 hours, most preferably,at least 60 hours. The temperature during disruption is typically keptlow, preferably at 4° C.

The insoluble aggregates that include one or more of the polypeptides ofthe present invention may be isolated by methods that are routine andknown to the art. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of outer membrane polypeptidesthat are insoluble in detergents requires centrifugal forces of at least50,000×g, typically 100,000×g. The use of such centrifugal forcesrequires the use of ultracentrifuges, and scale-up to process largevolumes of sample is often difficult and not economical with these typesof centrifuges. The methods described herein provide for the productionof insoluble aggregates large enough to allow the use of significantlylower centrifugal forces (for instance, 46,000×g). Methods forprocessing large volumes at these lower centrifugal forces are availableand known to the art. Thus, the insoluble aggregates can be isolated ata significantly lower cost.

Optionally and preferably, the sarcosine is removed from the isolatedpolypeptides. Methods for removing sarcosine from the isolatedpolypeptides are known to the art, and include, for instance,diafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, or affinity chromatography, and ultra filtration andwashing the polypeptides in alcohol by diafiltration. After isolation,the polypeptides suspended in buffer and stored at low temperature, forinstance, −20° C. or below.

Polypeptides of the present invention may also be isolated from microbesusing methods that are known to the art. The isolation of thepolypeptides may be accomplished as described in, for instance, Emery etal., (U.S. Pat. No. 5,830,479) and Emery et al., (U.S. PatentApplication US 20030036639 A1).

In those aspects of the present invention where a whole cell preparationis to be made, after growth a microbe can be killed with the addition ofan agent such as glutaraldehyde, formalin, or formaldehyde, at aconcentration sufficient to inactivate the cells in the culture. Forinstance, formalin can be added at a concentration of 3% (vol:vol).After a period of time sufficient to inactivate the cells, the cells canbe harvested by, for instance, diafiltration and/or centrifugation, andwashed.

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. The animal can be, for instance, avian (including,for instance, chickens or turkeys), bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), a companion animal (including, forinstance, horses), members of the family Cervidae (including, forinstance, deer, elk, moose, caribou and reindeer), or human.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks,after the first administration of the composition. Subsequent boosterscan be administered one, two, three, four, or more times annually.Without intending to be limited by theory, it is expected that in someaspects of the present invention annual boosters will not be necessary,as an animal will be challenged in the field by exposure to microbesexpressing polypeptides present in the compositions having epitopes thatare identical to or structurally related to epitopes present onpolypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for inducing theproduction of antibody in an animal or by recombinant techniques. Theantibody produced includes antibody that specifically binds at least onepolypeptide present in the composition. In this aspect of the invention,an “effective amount” is an amount effective to result in the productionof antibody in the animal. Methods for determining whether an animal hasproduced antibodies that specifically bind polypeptides present in acomposition of the present invention can be determined as describedherein.

The method may be used to produce antibody that specifically bindspolypeptides expressed by a microbe other than the microbe from whichthe polypeptides of the composition were isolated. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts with the epitope of the antigen that induced the synthesis ofthe antibody, or interacts with a structurally related epitope. At leastsome of the polypeptides present in the compositions of the presentinvention typically include epitopes that are conserved in thepolypeptides of different species and different genera of microbes (seeExample 26). Accordingly, antibody produced using a composition derivedfrom one microbe is expected to bind to polypeptides expressed by othermicrobes and provide broad spectrum protection against gram negativeorganisms. Examples of gram negative microbes to which the antibodyspecifically binds are enteropathogens, for instance, members of thefamily Enterobacteriaceae.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in animals that maybe caused by, or associated with, a microbe. Such conditions include,for instance, gram negative microbial infections. Examples of conditionscaused by microbial infections include mastitis, intestinal colonizationby a microbe, metritis, strangles, intrauterine infections, odemadisease, enteritis, chronic reproductive infections, laminitis,mastitis, and acute or chronic chlamydiosis, colibacillosis,ehrlichiosis, leptospirosis, pasteurellosis, pseudotuberculosis, andsalmonellosis. Examples of conditions that may be caused by microbialinfections include performance characteristics such as decreased milkproduction, high somatic cell counts, poor milk quality, and weightloss. Treatment of these conditions can be prophylactic or,alternatively, can be initiated after the development of a conditiondescribed herein. Treatment that is prophylactic, for instance,initiated before a subject manifests symptoms of a condition caused by amicrobe, is referred to herein as treatment of a subject that is “atrisk” of developing the condition. Typically, an animal “at risk” ofdeveloping a condition is an animal present in an area where thecondition has been diagnosed and/or is likely to be exposed to a microbecausing the condition. Accordingly, administration of a composition canbe performed before, during, or after the occurrence of the conditionsdescribed herein. Treatment initiated after the development of acondition may result in decreasing the severity of the symptoms of oneof the conditions, or completely removing the symptoms. In this aspectof the invention, an “effective amount” is an amount effective toprevent the manifestation of symptoms of a disease, decrease theseverity of the symptoms of a disease, and/or completely remove thesymptoms. The potency of a composition of the present invention can betested according to established standard methods detailed at Title 9 ofthe Code of Federal Regulations, section 113. For instance, 9 CFR §113.120(c) and 9 CFR § 113.123(c) describe standard methods fordetermining the potency of the composition against a standard referencebacterin of Salmonella enterica serovar Typhimurium and Salmonellaenterica serovar Dublin, respectively. Methods for determining whetheran animal has the conditions disclosed herein and symptoms associatedwith the conditions are routine and known to the art (see, for instance,Zhang et al., Infect. Immun., 71:1-12 (2003)).

In one aspect the invention is also directed to treating a gram negativemicrobial infection in an animal in an animal. The method includesadministering an effective amount of a composition of the presentinvention to an animal having or at risk of having a gram negativeinfection, and determining whether at least one symptom of the gramnegative infection is reduced. The successful treatment of a gramnegative microbial infection in an animal is disclosed in Examples 3-9.Working Examples 3-5, 6, and 9 demonstrate the protection againstdisease by caused by Salmonella enterica serovar Newport and bySalmonella enterica serovar Dublin in mouse models by administering acomposition of the present invention. Working Examples 7-8 demonstratethe protection against disease by caused by E. coli O157:H7 in a mousemodel by administering a composition of the present invention. Thesemouse models are a commonly accepted model for the study of humandisease caused by these microbes.

The present invention is also directed to decreasing colonization of theintestinal tract or reproductive tract of an animal by a gram negativemicrobe. The method includes administering an effective amount of acomposition of the present invention to an animal colonized by, or atrisk of being colonized by a gram negative microbe. In this aspect ofthe invention, an “effective amount” is an amount effective to decreasecolonization of the animal by the microbe. Colonization of an animal'sintestinal tract by a microbe can be determined by measuring thepresence of the microbe in the animal's feces. The successful decreaseof colonization by Salmonella and by E. coli is disclosed in Examples 6,7-8, 10-15, and 16-19. Working Examples 6 and 10-15 demonstrate thedecreased colonization by Salmonella in mice and in cattle. WorkingExamples 7-8 and 16-19 demonstrate the decreased colonization by E. coliO157:H7 in mice and in cattle. Cattle are considered to be one of theimportant natural reservoirs of E. coli O157:H7 that contaminate foodand cause human disease. Methods for evaluating the colonization of ananimal's reproductive tract by a microbe are routine and known to theart. It is expected that decreasing the colonization of an animal by amicrobe will reduce transmission of the microbe to humans.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare antibody compositions from serum, plasma, blood,colostrum, etc. for passive immunization therapies. Antibodycompositions comprising monoclonal antibodies and/or anti-idiotypes canalso be prepared using known methods. Passive antibody compositions andfragments thereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods for later use in a concentrated or reconstituted form suchas, for instance, lavage solutions, impregnated dressings and/or topicalagents and the like. Passive immunizing preparations may be particularlyadvantageous for treatment of acute systemic illness, or passiveimmunization of young animals that failed to receive adequate levels ofpassive immunity through maternal colostrum. Antibodies useful forpassive immunization may also be useful to conjugate to various drugs orantibiotics that could be directly targeted to bacteria expressing theseproteins during a systemic or localized infection.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically bind polypeptides of the presentinvention, and diagnosing whether an animal may have a condition causedby a microbe expressing polypeptides described herein, or expressingpolypeptides that share epitopes with the polypeptides described herein.Such diagnostic systems may be in kit form. The methods includecontacting an antibody with a preparation that includes polypeptides ofthe present invention to result in a mixture. The antibody may bepresent in a biological sample, for instance, blood, milk, or colostrum.The method further includes incubating the mixture under conditions toallow the antibody to specifically bind the polypeptide to form apolypeptide:antibody complex. As used herein, the term“polypeptide:antibody complex” refers to the complex that results whenan antibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides of the present invention may also includereagents, for instance a buffer, that provide conditions appropriate forthe formation of the polypeptide:antibody complex. The polypeptide:antibody complex is then detected. The detection of antibodies is knownin the art and can include, for instance, immunofluorescence andperoxidase. The methods for detecting the presence of antibodies thatspecifically bind to polypeptides of the present invention can be usedin various formats that have been used to detect antibody, includingradioimmunoassay and enzyme-linked immunosorbent assay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The kitincludes at least one of the polypeptides of the present invention, or anumber of polypeptides that is an integer greater than 1 (e.g., at least2, at least 3, etc.), in a suitable packaging material in an amountsufficient for at least one assay. Optionally, other reagents such asbuffers and solutions needed to practice the invention are alsoincluded. Instructions for use of the packaged polypeptides are alsotypically included. As used herein, the phrase “packaging material”refers to one or more physical structures used to house the contents ofthe kit. The packaging material is constructed by well known methods,generally to provide a sterile, contaminant-free environment. Thepackaging material has a label which indicates that the polypeptides canbe used for detecting antibody that specifically binds polypeptides ofthe present invention. In addition, the packaging material containsinstructions indicating how the materials within the kit are employed todetect the antibody. As used herein, the term “package” refers to asolid matrix or material such as glass, plastic, paper, foil, and thelike, capable of holding within fixed limits the polypeptides. Thus, forexample, a package can be a microtiter plate well to which microgramquantities of polypeptides have been affixed. “Instructions for use”typically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1 Production and Isolation of Metal Regulated Proteins

Gram negative enteric bacteria belonging to the familiesEnterobacteriaceae and Vibrionaceae as well as other gram negativebacteria can be grown under controlled fermentation conditions so as toexpress proteins, including proteins associated with the outer membrane.The bacteria can be harvested and the proteins can then be isolated andused as immunogens in a composition described in detail in the followingexample.

The immunizing compositions used in the following examples were preparedusing the proteins derived from two enteric pathogens; a multi-drugresistant isolate of Salmonella enterica serovar Newport and anEscherichia coli O157:H7 isolate, both originating from a bovinespecies.

Salmonella enterica serovar Newport was isolated from fecal cultures ofcows at a commercial dairy showing clinical signs of salmonellosis. Thisisolate was also recovered from the owners of the dairy who became sickafter ingesting raw milk. Isolation was accomplished using BrilliantGreen Sulfa broth and Brilliant Green Selective agar plates. The isolatewas serotyped by the Centers for Veterinary Biologics Laboratory (Ames,Iowa) and by the Minnesota Poultry Testing Laboratory, Minnesota Boardof Animal Health (Willmar, Minn.). The Escherichia coli isolate O157:H7originated from steers isolated from a commercial feed lot. As detectedby PCR, the strin was found to be serotype O157:H7, and possessed theeaeA and hlyA genes but not the stx1 and stx2 genes.

Master seed stocks of the Salmonella Newport and E. coli O157:H7isolates were prepared by inoculating each of the isolates into 200 mlof Tryptic Soy Broth (Difco Laboratories, Detroit, Mich.) containing 50micrograms per milliliter (μg/ml) of 2,2-dipyridyl (Sigma-Aldrich St.Louis, Mo.). The cultures were grown while stirring at 200 rpm for 6hours at 37° C. The bacteria were collected by centrifugation at10,000×g. The bacterial pellets from each isolate was resuspended into100 ml physiological saline (0.85%) containing 20% glycerol, andsterilely dispensed into 2 ml cryogenic vials (1 ml per vial) and storedat −90° C. Each isolate was given an identification number designatingit as a master seed. The master seed number for Salmonella Newport wasMS020508 while the O157:H7 isolate was designated as BEcO157(stx-). Eachmaster seed was expanded into a working seed that was then used for theproduction of metal regulated proteins. A large-scale production processwas developed involving fermentation, bacterial harvest, disruption,solubilization, concentration, diafiltration, and isolation of finalproduct.

Fermentation

A cryogenic vial of the working seed (1 ml at 10⁹ CFU/ml) was used toinoculate 500 ml of 37° C. tryptic soy broth (TSB) without dextrose(Difco) containing 50 micrograms 2,2-dipyridyl (Sigma), 2.7 grams BiTekyeast extract (Difco) and glycerol (3% vol/vol). The culture wasincubated at 37° C. for 12 hours while stirring at 200 rpm, and thenadded to 2 liters of the above media. This second culture was allowed togrow for an additional 4 hours at 37° C. This culture was used toinoculate a 20-liter Virtis bench-top fermentor, (Virtis, Gardiner,N.Y.) charged with 13 liters of the above-described media. The pH washeld constant between 6.9 and 7.1 by automatic titration with 30% NaOHand 10% HCL. The stirring speed was adjusted to 400 revolutions perminute (rev/minute), and the culture aerated with 11 liters air/minuteat 37° C. Foaming was controlled automatically by the addition of 11 mldefoamer (Mazu DF 204 Chem/Serv, Minneapolis, Minn.). The culture wasallowed to grow continuously at these conditions for 4 hours at whichtime was sterilely pumped into a 150-liter fermentor (W. B. Moore,Easton, Pa.). The fermentor was charged with 115 liters TSB withoutdextrose (3,750.0 grams), BiTek yeast extract (625 grams), glycerol(3750 ml), 2,2-dypyrdyl (3.13 grams) and Mazu DF 204 defoamer (100 ml).The parameters of the fermentation were as follows: dissolved oxygen(DO) was maintained at 30%+/−10% by increasing agitation to 220rev/minute sparged with 60 liters of air/minute and 10 pounds per squareinch (psi) back pressure. The pH was held constant between 6.9 and 7.1by automatic titration with 30% NaOH and 10% HCL. The temperature wasmaintained at 37° C. At hour 4.5 (optical density 8-9 at 540nanometers), the culture was supplemented with additional nutrients byfeeding 7 liters of media containing 1,875 grams TSB without dextrose,313 grams yeast extract 3.13 grams 2,2-dipyridyl and 1,875 ml ofglycerol. The rate of feed was adjusted to 29 ml/minute while increasingagitation to 675 rpm. At the end of the feed (hour 8.5) the fermentationwas allowed to continue for an additional three hours at which point thefermentation was terminated by lowing the temperature of the fermentorto 10° C. (optical density 35-40 at 540 nanometers at a 1:100 dilution).The culture was sterilely transferred to a 200-liter tank (LEE ProcessSystems and Equipment model 2000LDBT) in preparation for harvest.

Harvest

The bacterial fermentation was concentrated and washed using a PallFiltron Tangential Flow Maxiset-25 (Pall Filtron Corporation, Northboro,Mass.) equipped with two 30 ft² Alpha 300-K open channel filters,catalog No. AS30005, (Pall Filtron) connected to a Waukesha Model U-60feed pump (Waukesha Cherry-Burrell, Delevan, Wis.) The original culturevolume of 125 liters was reduced to 25 liters (2.5 liters/minute) usinga filter inlet pressure of 15 psi and a retentate pressure of 0 psi. Thebacterial retentate was adjusted back up to 50 liters usingphysiological saline (0.85%) and then concentrated again to 15 liters tohelp remove any contaminating exogenous proteins, etc. The retentate (15liters) was adjusted to 35 liters using sterile Osmotic Shock Buffer(OMS) containing 7.26 grams/liter Tris-base and 0.93 grams/liter EDTAadjusted to a pH of 8.5. The EDTA in the OMS serves to remove much ofLPS from the cell wall, while the elevated pH prevents much of theproteolytic degradation after freezing and disruption. Proteaseinhibitors may be used instead of, or in addition to, an elevated pH.The retentate was mixed thoroughly while in the 200-liter tank using abottom mount magnetically driven mixer. The retentate was sterilelydispensed (3.5 liters) into sterile 4 liter Nalgene containers No. 2122and placed into a −20° C. freezer for storage. Freezing the bacterialpellet serves to weaken the cell wall structure making downstreamdisruption more efficient. The pellet mass was calculated bycentrifuging 30 ml samples of the fermented culture and final harvest.Briefly, pre-weighted 50 ml Nalgene conical tubes were centrifuged at39,000×g for 90 minutes in a Beckman J2-21 centrifuge using a JA-21rotor (Beckman Instruments, Palo Alto Calif.). At the end of the run,the supernate was poured off and the tubes were weighed again. Thepellet mass was calculated for each stage. The fermentation processyielded a wet pellet mass of 9.0 kilograms.

Disruption (Homogenization)

Twenty kilograms of frozen bacterial cell slurry in OMS were thawed at4° C. (20 kg of pellet mass). The liquid culture suspension from eachcontainer was aseptically aspirated into a steam in place 250 literjacketed process tank (Lee, Model 259LU) with a top mounted mixer(Eastern, Model TME-1/2, EMI Incorporated, Clinton, Conn.) containing222 liters OMS pH 8.5 containing 0.1 grams thimerosal/liter aspreservative. The volume of OMS was determined by dividing the pelletmass (in grams) by 900 and then multiplying the result by 10 to get thehomogenizing volume in liters (gram pellet mass/900×10=litershomogenizing volume). The bulk bacterial suspension was chilled to 4° C.with continuous mixing for 18 hours at 200 rpm at which time it wasdisrupted by homogenization. Briefly, the 250 liter tank containing thebacterial suspension was connected to a model 12.51 H RannieHomogenizer, (APV Systems, Rosemont, Ill.). A second 250 liter jacketedprocess tank (empty) was connected to the homogenizer such that thefluid in the process tank could be passed through the homogenizer, intothe empty tank and back again, allowing for multiple homogenizing passeswhile still maintaining a closed system. The temperature duringhomogenization was kept at 4° C. At the start of each pass, fluid wascirculated at 70 psi via a Waukesha model 10DO pump (Waukesha) throughthe homogenizer (160 gallons/hour) and back to the tank of origin, whilethe homogenizer pressure was adjusted to 13,500 psi. Prior to the firstpass, two pre-homogenizing samples were withdrawn from the homogenizerto establish a baseline for determining the degree of disruption andmonitoring of pH. The degree of disruption was monitored bytransmittance (% T at 540 nm at 1:100 dilution) compared to thenon-homogenized sample. The number of passes through the homogenizer wasstandardized for different organisms based on the integrity of the cellwall and variation in the degree of disruption, which had a directcorrelation in the efficiency of solubilization and quality of endproduct. For example, the disruption of Salmonella passed three timesthrough the homogenizer gave a final percent transmittance between78-83% T at a 1:100 dilution. E. coli having the same pellet mass andstarting OD gave a % T of 86-91% (at a 1:100 dilution) after the thirdpass. It has been observed that bacteria differ in their cell wallintegrity and vary in their capacity of disruption under identicalcondition. This variation can effect the degree and efficiency ofsolubilization and recovery of metal regulated proteins. In general,cells were passed through the homogenizer until the transmittance didnot increase after an additional pass.

After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30,Chem/Serv) was aseptically added to the homogenized bacterial suspensionfor solubilization. The amount of Sarcosine (30%) added equaled 0.0664times the solubilizing volume, in liters, (1.0 gram sarcosine/4.5 gramspellet mass). The tank was removed from the homogenizer and put onto achiller loop at 4° C. and mixed at 240 rpm for 60-70 hours. This timeperiod was important for complete solubilization. It was discovered thatincreasing the solubilization time in OMS at an elevated pH (8.0-8.5)that metal regulated proteins aggregated together forming largeinsoluble aggregates that were easily removed by centrifugation. Theoptimal OD after solubilization was usually between 25-30% T at 540 nm.

Protein Harvest

The aggregated metal regulated proteins within the solubilized processfluid were collected by centrifugation using T-1 Sharples, (Alfa LavalSeperations, Warminster, Pa.). Briefly, the tank of solubilizedhomogenate was fed into six Sharples with a feed rate of 250 ml/minuteat 17 psi at a centrifugal force of 46,000×g. The effluent was collectedinto a second 250 liter jacketed process tank through a closed sterileloop allowing for multiple passes through the centrifuges whilemaintaining a closed system. The temperature during centrifugation waskept at 4° C. The solubilized homogenate was passed 8 times across thecentrifuges. Fifty percent of the protein was collected after the secondpass, at which point the solubilized fluid was concentrated to ⅓ of itsoriginal volume. This decrease in volume shortened the process time forthe next 6 passes. Briefly, the solubilized homogenate tank wasaseptically disconnected from the centrifuges and connected to aMillipore Pellicon Tangential Flow Filter assembly (MilliporeCorporation, Bedford, Mass.), equipped with a 25 ft² screen-channelseries Alpha 10K Centrasette filter (Pall Filtron) connected to aWaukesha Model U30 feed pump for concentration. After concentration,centrifugation was continued until the process was completed. Proteinwas collected after each pass. The protein was collected, resuspendedand dispensed in 50 liters Tris-buffer pH 8.5 containing 0.3% formalin(Sigma) as preservative.

Diafiltration

The protein suspension was washed by diafiltration at 4° C. to removeany contaminating sarcosine that may have been bound to the protein.Briefly, the 50 liters of protein was sterilely aspirated into a 200liter process tank containing 50 liters sterile Tris-buffer, pH 8.5,equipped with a bottom mount Dayton mixer, Model 2Z846 (Dayton Electric,Chicago, Ill.) rotating at 125 rev/minute. The process tank wassterilely connected to a Millipore Pellicon Tangential Flow Filterassembly (Millipore Corporation), equipped with a 25 ft² screen-channelseries Alpha 10K Centrasette filter (Pall Filtron) connected to aWaukesha Model U30 feed pump. The 100 liter protein solution wasconcentrated by filtration to a target volume of 5.45 times the proteinpellet mass, at which point Tris-buffer, pH 7.4, containing 5% isopropylalcohol was slowly added to the concentrate from a second process tank.Isopropyl alcohol is believed to cause a slight unfolding of the proteinstructure allowing for the removal of bound sarcosine withoutcompromising the immunogenicity of the protein. Diafiltration continueduntil the pH stabilized to 7.4 at which point 50 liters Tris-buffer pH7.4 was slowly added by diafiltration to remove residual alcohol. Theprotein suspension was then concentrated to approximately 25 liters. Theprotein concentrate was aseptically dispensed (3.5 liters) into sterile4 liter Nalgene containers and placed into a −20° C. freezer forstorage.

This process produces a composition containing metal regulated proteinswith a decrease in the amount of LPS and very little to no sarcosineresidue. The protein was examined by SDS-PAGE for purity and bandingprofile, and also examined for bacterial contamination, residualsarcosine and LPS. The banding profile of the finished product showedconsistent patterns as examined by electrophoresis. The composition wastested for sarcosine by the use of a modified agar gel diffusion test inwhich sheep red blood cells (5%) were incorporated into an agar base(1.5%). Wells were cut into the agar and samples of the finished productalong with control samples of known concentrations of sarcosine at 0.05,0.1, 0.2, 0.3, 0.4, 0.5 1.0 and 2.0% were placed into the wells. The gelwas incubated at 25° C. for 24 hours and the degree of hemolysis wasdetermined compared to the controls. The process removes the level ofdetectable sarcosine below 0.05%, which at this concentration showedminimal hemolysis in control samples. The concentration of LPS isexamined by a Limulus amebocyte lysate (LAL) test available under thetradename PYROTELL (Associates of Cape Cod, Inc., East Falmouth, Mass.).

The compositions used in the following examples were prepared andharvested as described in Example 1. The efficacy of each compositionwas evaluated in separate experiments based on the route of challenge.Data was collected on the following parameters; 1) the potency of theimmunizing compositions, which was evaluated against a live virulentchallenge given intraperitoneally to measure systemic protection, 2) theefficacy of each composition, which was evaluated after administeringthe challenge dose orally to evaluate the effect on colonization orfecal shedding of the challenge organism, and 3) examination of theinjection sites for any adverse tissue reaction.

Example 2 Preparation of the Immunizing Compositions Derived from S.enterica Serovar Newport and E. coli O157:H7

The proteins made from S. enterica serovar Newport and E. coli asdescribed in Example 1 were used to prepare two compositions foradministration to animals. The composition prepared from S. entericaserovar Newport contained the proteins described in Table 2, and thecomposition prepared from E. coli O157:H7 contained the proteinsdescribed in Table 6. A stock vaccine was prepared from each compositionby emulsifying the aqueous protein suspension (500 μg total protein/ml)into the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston,Nebr.) using an IKA Ultra Turrax T-50 homogenizing vessel (IKA,Cincinnati, Ohio). The stock vaccine was used at two differentinjectable volumes depending on the target animal it would be used in. Amouse dose was administered to give a final dose of 50 μg total proteinin a 0.1 ml injectable volume with an adjuvant concentration of 22.5%vol/vol. The bovine dose was given using a two milliliter injectablevolume to provide a dose of 1000 μg total protein. A placebo wasprepared by replacing the antigen with physiological saline in the aboveformulation and emulsifying the suspension into EMULSIGEN to give andadjuvant concentration of 22.5%.

A standard reference bacterin of Salmonella enterica serovar Dublin wasobtained from the Center of Veterinary Biologics-Laboratory (UnitedStates Department of Agriculture, Ames, Iowa, strain APHIS NVSL #82,Salmonella Dublin, Lot Number IRP DSC #5) for use as a control referencein accordance with the standardized mouse model described in 9 CFR113.123. This was provided as a whole cell bacterin prepared in AlOH.

Example 3 Mouse Vaccination and Challenge Study (Systemic Evaluation)

The efficacy of the S. enterica serovar Newport vaccine was carried outagainst a live virulent challenge in mice. One hundred twenty five(N=125) female CF-1 mice obtained from Harlan Breeding Laboratories(Indianapolis, Ind.) weighing 16-22 grams were equally distributed intofive groups (25 mice/group). Mice were housed in polycarbonate mousecages (Ancore Corporation, Bellmore, N.Y.). Two cages were used for eachtreatment group to minimize the number of mice for each cage. Groupswere designated as 1-5. Food and water were supplied ad libitum to allmice.

The potency of the vaccine was tested at four different concentrations;non-diluted stock vaccine (Group 1, 50 μg/0.1 ml), 1:10 (volume diluent:volume protein solution) (Group 2, 5.0 μg/0.1 ml), 1:100 (Group 4, 0.5μg/0.1 ml), 1:1000 (Group 4, 0.05 μg/0.1 ml) and a Placebo(non-vaccinated/challenged control group) (Group 5). EMULISIGEN was usedas the diluent for diluting the stock vaccine so as to maintain theconcentration of adjuvant at a 22.5% for each treatment group. Mice werevaccinated intraperitoneally two times at 14 day intervals. The volumeadministered was 0.1 ml/mouse.

Example 4 Preparation of Challenge Organism

The S. enterica serovar Newport as described above was used forchallenge. Briefly, the isolate from a frozen stock was streaked onto ablood agar plate and incubated at 37° C. for 18 hours. A single colonywas subcultured into 50 ml Tryptic Soy Broth (Difco) containing 25 μg/ml2, 2′ dipyridyl. The culture was incubated at 37° C. for 6 hours whilerotating at 200 rpm, at which point was centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellet was washedtwice by centrifugation in physiological saline at 4° C. The finalpellet was resuspended in 25 ml of physiological saline and used forchallenge. Just prior to challenge, lml of the above bacterialsuspension was serially diluted ten fold to enumerate the number ofCFU/mouse dose.

Example 5 Challenge

Fourteen days after the 2nd vaccination, mice in groups 1-5 wereintraperitoneally challenged with 0.2 ml of S. enterica serovar Newportat 7.6×10⁸ colony forming units (CFU) prepared as described at example4. Mortality was recorded daily for 14 days after challenge. The resultsshowed a strong protective index that correlated with dilution of thevaccine (Table 18). Twenty five (100%) of the non-vaccinated mice (Group5) died within 14 days after challenge. In contrast, only 1 mouse (4.0%)died given the non-diluted vaccine of Group 1. Mortality increased witheach 10 fold serial dilution as seen in FIG. 1 (Group 2, 8.0%), (Group3, 48.0%) and (Group 4, 80.0%). The vaccine showed a high degree ofsystemic protection as compared to non-vaccinated mice of Group 5(Placebo). The vaccine prepared from S. enterica serovar Newport washighly efficacious in preventing mortality associated with a lethal S.enterica serovar Newport challenge.

TABLE 18 Mortality of Vaccinated and Non-Vaccinated Mice Following IPChallenge with S. enterica serovar Newport Groups # Mice # Dead Percentmortality (%) Group 1 (non-diluted) 25  1/25 4.3 Group 2 (1:10) 25  2/258.3 Group 3 (1:100) 25 12/25 48.0 Group 4 (1:1000) 25 15/25 88.0 Group 5(non-vaccinated/ 25 25/25 100.0 challenged)

Example 6 Mouse Vaccination and Oral Challenge Study with S. entericaSerovar Newport (Evaluation of Fecal Shedding)

In this experiment the efficacy of the S. enterica serovar Newportvaccine was carried out against a live oral challenge in mice. Theoutcome parameters used to evaluate vaccine efficacy in this experimentwere 1) individual mouse mortality, and 2) differences in theconcentration of Salmonella being shed between treatment groups afterchallenge. Twenty (N=20) female CF-1 mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into two groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.). Twocages were used, one for each treatment group. Groups were designated asplacebo, non-vaccinated (Group 1) and vaccinated (Group 2). Food andwater were supplied ad libitum to all mice.

Mice were vaccinated three times at 14 day intervals subcutaneously withthe placebo and/or the S. enterica serovar Newport vaccines of Example2. The volume of vaccine administered was 0.1 ml/mouse. Seven days afterthe third vaccination, mice in groups 1 and 2 were orally challengedwith S. enterica serovar Newport at 2.8×10⁸ colony forming units (CFU)in a volume of 0.2 cc. The challenge organism was prepared as describedin example 4.

To enumerate the difference in fecal shedding between the control andvaccinated groups, mouse droppings were collected at 12, 24, 36 and 48hours post challenge. Droppings were collected by placing a sterile padon the floor of each cage 1 hour prior to collection. At each timeperiod the pad was removed and placed into a laminar flow hood. Usingsterilely flamed forceps, twenty individual droppings were randomlycollected. The forceps were flamed between each collection so as not tocross-contaminate samples. Individual droppings were placed into sterilesaline dilution blanks (0.9 ml), two droppings per tube to give tentubes. Each sample was macerated using a sterile 1 ml pipette andserially diluted 10 fold. Dilutions were plated on Brilliant GreenSulfur Agar (Difco Laboratories, Detroit, Mich.) incubated at 37 C for48 hours. The number of bacteria was enumerated for each sample and thelog₁₀ colony forming units were averaged for each treatment group ateach time period.

Table 19 shows the difference in the fecal shedding between vaccinatedand non-vaccinated mice after an oral challenge with S. enterica serovarNewport at each time period. There was a large difference betweentreatment groups in the amount of Salmonella shedding in fecespost-challenge. The challenge dose represented as time 0 in Table 4shows the initial inoculum given to each mouse. Within twelve hours postchallenge there was a dramatic decrease in the amount of Salmonellabeing shed from the vaccinated group as compared to the Placebo group.Averaged across the study period and accounting for repeated estimates,vaccinates shed less Salmonella at each sampling period when compared tonon-vaccinates, with a degree of significance of P=0.02. The amount ofSalmonella being shed in the vaccinated group dramatically declined witheach sampling period as compared to the non-vaccinated Placebo group(FIG. 2). At 48 hours post challenge the difference in the amount ofSalmonella being shed between the vaccinated and non-vaccinated groupwas greater then 4 log CFU (Table 19, FIG. 2).

TABLE 19 The Difference in Shedding of Salmonella Newport Between theNon- Vaccinated and Vaccinated Treatment Groups after Oral Challenge.Mean log₁₀ Colony Forming Units Sampling Times Group 1 (Non-vaccinated)Group 2 (Vaccinated) Challenge Dose 8.4 8.4 (time 0) 12 hours 5.5 3.1 24hours 4.2 0.8 36 hours 3.7 0.43 48 hours 5.0 0.16

At the 12 hour sampling period three mice (30%) died in the vaccinatedgroup with no further mortality occurring within 14 days afterchallenge. Nevertheless, in the non-vaccinated Placebo group three micedied within 12 and 24 hours and 4 mice died between 48 and 56 hours (70%total). It's interesting to note that mortality seemed to be directlycorrelated with the amount of Salmonella being shed. This is illustratedin the vaccinated group where mortality occurred at an early stage wherethe level of Salmonella being shed was high (Table 19, FIG. 2). Thisobservation was observed in both groups where three mice died within 24hours while the amount of Salmonella being shed was high. However, asthe incidence of shedding declined in the vaccinated group so didmortality. In contrast, as the incidence of shedding increased in thePlacebo as seen at 48 hours, it appeared to be directly correlated withmortality, since 4 mice died within this time period.

Due to the unequal distribution in the number of mice between groupsafter 48 hours no further sampling was carried out beyond this timeperiod. Nevertheless, the results clearly demonstrate that subcutaneousvaccination with the composition can prevent colonization by Salmonella.In addition, the secondary sequelae due to systemic infection was alsoinhibited as seen in the difference in mortality between the two groups.

Example 7 Mouse Vaccination and Oral Challenge Study with Escherichiacoli O157:H7 (Evaluation of Fecal Shedding)

In this experiment, the efficacy of the Escherichia coli O157:H7 vaccineof example 2 was carried out against a live oral challenge in mice. Theoutcome parameter used to evaluate the efficacy of the vaccine in thisexperiment was to enumerate differences in the concentration of thechallenge organism being shed between treatment groups after challenge.Twenty (N=20) female CF-1 mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into two groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.). Twocages were used, one for each treatment group. Groups were designated asplacebo, non-vaccinated (Group 1) and vaccinated (Group 2). Food andwater were supplied ad libitum to all mice.

Mice were vaccinated three times at 14 day intervals subcutaneously withthe placebo and/or the E. coli O157:H7 vaccines of Example 2. The volumeof vaccine administered was 0.1 ml/mouse.

Example 8 Preparation of E. coli O157:H7 Challenge Organism

To enhance the isolation rate of the E. coli O157:H7 challenge organismfrom fecal samples the isolate was made nalidixic acid resistant.Briefly, the isolate from a frozen stock was streaked onto EosinMethylene Blue (EMB) agar plate and incubated at 37° C. for 18 hours. Asingle colony was subcultured into 50 ml Tryptic Soy Broth (Difco)containing 25 μg/ml 2, 2′ dipyridyl. The culture was incubated at 37° C.for 6 hours while rotating at 200 rpm, at which point was subculturedinto Tryptic Soy Broth containing 25 μg/ml 2,2 dipyridyl and 100μg/mlnalidixic acid and again incubated at 37° C. for 18 hours. Onehundred microliters of the above culture containing approximately 10⁸CFU/ml viable organisms was spread over the surface of an EMB agar platecontaining 500 μg nalidixic acid. The plates were incubated at 37° C.for 48 hours and the colonies that grew were cloned by plating on EMBcontaining 100 μg/ml nalidixic acid. A number of nalidixic acidresistant colonies were amplified by sub-culturing into TSB containing25 μg/ml 2,2 dipyridyl and 100 μg/ml nalidixic acid. A stable nalidixicacid resistant isolate was selected as the challenge organism bysub-culturing the isolate continuously in TSB containing 100 μg/mlnalidixic acid to enhance the stability of the organism. The outermembrane protein profile of the nalidixic acid resistant isolateexpressed identical banding profiles as the parent wild type grown underiron-restriction having molecular weights of 89 kDa, 85 kDa, 81 kDa, 78kDa and porins at 36-39 kDa. The nalidixic acid resistant isolate of E.coli O157:H7 was maintained as a frozen stock culture in TSB containing25 μg/ml 2,2 dipyridyl, 100 μg/ml nalidixic acid and 20% glycerol storedat −90° C.

The nalidixic acid resistant isolate of E. coli O157:H7 as describedabove was used for challenge. The isolate from the frozen stock wassub-cultured onto an EMB agar plate containing 150 μg/mlnalidixic acidand incubated at 37° C. for 18 hours. A single colony was subculturedinto 50 ml Tryptic Soy Broth (Difco) containing 25μ/ml 2, 2′ dipyridyland 250 μg nalidixic acid. The culture was incubated at 37° C. for 6hours while rotating at 200 rpm, at which point was centrifuged at10,000×g for 10 minutes at 4° C. to pellet the bacteria. The bacterialpellet was washed twice by centrifugation in physiological saline at 4°C. The final pellet was resuspended in 25 ml of physiological salinecontaining 250 μg nalidixic acid and used for challenge. Just prior tochallenge, 1 ml of the above bacterial suspension was serially dilutedten fold to enumerate the number of CFU/mouse dose.

Seven days after the third vaccination, mice in groups 1 and 2 wereorally challenged with 0.2 ml of the nalidixic acid resistant isolate ofE. coli at 2.0×10⁸ CFU.

To enumerate the difference in fecal shedding between the control andvaccinated groups, mouse droppings were collected at 12, 24, 36, 48, 56and 72 hours post challenge. Droppings were collected as before byplacing a sterile pad on the floor of each cage 1 hour prior tocollection. At each time period the pad was removed and placed into alaminar flow hood. Using sterilely flamed forceps twenty individualdroppings were randomly collected. The forceps were flamed between eachcollection so as not to cross-contaminate samples. Individual droppingswere placed into sterile saline dilution blanks (0.9 ml), two droppingsper tube to give ten tubes. Each sample was macerated using a sterile 1ml pipette and serially diluted 10 fold. Dilutions were plated on EMBagar containing 150 μg nalidixic acid/ml incubated at 37 C for 48 hours.The number of bacteria was enumerated for each sample and the log₁₀colony forming units were averaged for each treatment group at each timeperiod.

Table 20 shows the difference in the fecal shedding between vaccinatedand non-vaccinated mice after an oral challenge with the nalidixic acidresistant isolate of E. coli. There was a significant difference in theamount of E. coli O157:H7 being shed between groups at each samplingperiod. The challenge dose represented as time 0 in Table 20 shows theinitial inoculum given to each mouse. Within twelve hours post challengethere was a dramatic decrease in the amount of E. coli O157:H7 beingshed from the vaccinated group as compared to the Placebo group with adegree of significance of P=0.03. The amount of E. coli O157:H7 beingshed in the vaccinated group dramatically declined with each samplingperiod as compared to the non-vaccinated mice (FIG. 3). Averaged acrossthe study period, vaccinates shed approximately 2 logs less E. coli whencompared to the non-vaccinated controls. At 56 and 72 hours postchallenge the shedding incidence of the challenge organism in thevaccinated group was undetectable as compared to the non-vaccinatedcontrols which continued to shed for the duration of the study period.

TABLE 20 The Difference in Shedding of E. coli O157:H7 Between the Non-Vaccinated and Vaccinated Treatment Groups after Challenge. Mean log₁₀Colony Forming Units Group 1 Group 2 Sampling Times (Non-vaccinated)(Vaccinated) Challenge Dose (time 0) 8.3 8.3 12 hours 6.2 5.1 24 hours3.9 1.7 36 hours 2.1 0.63 48 hours 3.2 0.1 56 hours 2.1 0 72 hours 1.2 0

Between the 12 and 24 hour sampling period two mice (20.0%) died in thevaccinated group with no further mortality occurring within 14 daysafter challenge. However, in the non-vaccinated Placebo group five mice(50.0%) died during the sampling period with no further mortalityoccurring after 72 hours or within the 14 day observation period.

These results demonstrate for the first time that a vaccine compositionas described herein can prevent the colonization and/or growth of E.coli O157:H7 through a subcutaneous vaccination as well as reducemortality due to the secondary sequelae from systemic infection.

Example 9 The Cross Protective Nature of Metal Regulated ProteinsAgainst a Homologous and Heterologous Salmonella Challenge in Mice

The vaccine Example 2 derived from S. enterica serovar Newport wasevaluated against a homologous and heterologous challenge using S.enterica serovar Dublin in a standardized mouse model as described inthe Code of Federal Regulations, Title 9, section 113.123.

Two hundred ten (N=210) female Harlan CF-1 mice obtained from HarlanBreeding Laboratories (Indianapolis, Ind.) weighing 16-22 grams wereequally divided into 9 treatment groups (20 mice/group) designated asgroups 1-9. The efficacy of each vaccine was tested at four differentconcentrations. The whole cell S. enterica serovar Dublin referencebacterin as described in example 2 was administered to four groupsdesignated as groups 1-4; Group 1 (non-diluted, 50 μg total protein),Group 2, (1:10 dilution, 5.0 μg total protein), Group 3, (1:100dilution, 0.5 μg total protein) and Group 4, (1:1000 dilution, 0.05 μgtotal protein). The S. enterica serovar Newport vaccine was alsoadministered at the same dilutions as described above in the same numberof mice, designated as groups 5-8 Group 5 (non-diluted), Group 6 (1:10),Group 7 (1:100) and Group 8 (1:1000) respectively. Group 9 was thecontrol group that was not vaccinated, but challenged. Since theSalmonella reference bacterin was prepared using whole cells andsupplied from an outside source the protein concentration was unknown.EMULISIGEN was used as the diluent for diluting the composition preparedusing MS020508 at a 22.5% concentration prepared in physiologicalsaline. The S. enterica serovar Dublin reference bacterin was dilutedusing phosphate buffered saline (PBS). All mice in groups 1-4 and 5-8were vaccinated with the appropriate vaccine intraperitoneally andrevaccinated 14 days after the first vaccination. The volumeadministered was 0.25 ml per mouse.

Fourteen days after the second vaccination, all mice in groups 1-9 wereintraperitoneally challenged with 9.8×10⁷ colony forming units (CFU) ofa virulent Salmonella enterica serovar Dublin isolate to evaluate thecross-protective nature of the S. enterica serovar Newport vaccineagainst a S. enterica serovar Dublin challenge (S. enterica serovarNewport vaccinated/S. enterica serovar Dublin challenged) compared tothe homologous group (S. enterica serovar Dublin vaccinated/S. entericaserovar Dublin challenged) The virulent Salmonella enterica serovarDublin isolate was obtained from The Center of VeterinaryBiologics-Laboratory (IRP SDC #5, United States Department ofAgriculture, Ames, Iowa). Mortality was recorded daily for 2 weekspost-challenge. Table 21 shows the percent mortality in mice following ahomologous and/or heterologous challenge with S. enterica serovarDublin.

TABLE 21 Mortality in mice following a homologous and/or heterologouschallenge with Salmonella Dublin Groups # Mice # Dead Percent mortality(%) Groups 5-8 (Reference Bacterin Vaccinated/Challenged with S.enterica serovar Dublin) Homologous Challenge Group 5 (non-diluted) 2010/20 50 Group 6 (1:10) 20 11/20 55 Group 7 (1:100) 20 16/20 80 Group 8(1:1000) 20 16/20 80 Group 9 (non-vaccinated) 20 20/20 100 Groups 1-4(S. enterica serovar Newport composition Vaccinated/ Challenged with S.enterica serovar Dublin) Heterologous Challenge Group 1 (non-diluted) 2010/20 50 Group 2 (1:10) 20 17/20 85 Group 3 (1:100) 20 20/20 100 Group 4(1:1000) 20 18/20 90

Twenty (100%) of the non-vaccinated mice (Group 9) died within 3 daysafter challenge (Table 21). Mice vaccinated with the composition derivedfrom S. enterica serovar Newport and challenged with S. enterica serovarDublin showed a high degree of cross-protection (Group 1) when comparedto mice vaccinated with the S. enterica serovar Dublin referencebacterin (Group 5). There was no difference in mortality between thesetwo groups. This data shows that the composition derived from S.enterica serovar Newport was protective against a live S. entericaserovar Dublin challenge as compared to the non-vaccinated control mice.Further, the composition derived from S. enterica serovar Newportprotected against a different serogroup of Salmonella showingheterologous protection: S. enterica serovar Newport is typed asserogroup C₂ whereas S. enterica serovar Dublin is a D₁ serogroup.

The results from this study provide strong evidence that the compositionincludes highly protective antigens that protect against a homologousand heterologous Salmonella challenge in mammals.

Example 10 Experimental S. enterica Serovar Newport Challenge in Calves

The purpose of this study was to evaluate the efficacy of the SalmonellaNewport vaccine described in Example 2 against a homologous S. entericaserovar Newport challenge in calves. The parameters used to evaluatevaccine efficacy were 1) individual calf morbidity as evidenced byrectal temperature, and 2) serological response to vaccination andquantitative enumeration of fecal shedding of S. enterica serovarNewport.

Thirty male Holstein steers (N=30) 4 to 6 months of age were randomlyassigned to two treatment groups, designated as Group 1 and Group 2.Group 1, which consisted of 20 steers, received the immunizingcomposition derived from S. enterica serovar Newport strain MS020508 asdescribed in Example 2. Steers in Group 2, which consisted of 10 steers(N=10), were vaccinated with a placebo (control group) made by preparingthe immunizing composition of example 2 without the addition of thecomposition derived from S. enterica serovar Newport. The antigen in thecontrol formulation was replaced with saline while keeping the adjuvantconcentration the same (22.5%). All calves in groups 1 and 2 werevaccinated with the appropriate vaccine subcutaneously and revaccinated21 days after the first vaccination.

Example 11 Blood and Fecal Sample Collection

Blood samples were collected from all steers on day 7 and again at 28,42 and 49 days after the first vaccination. The second vaccination was28 days after the first vaccination. All blood was collected in sterile13×75 mm vacutainer collection tubes, (SST No. 369783, Becton Dickinson,Franklin Lakes, N.J.). After clotting the blood tubes were centrifugedat 800×g for 30 minutes and frozen at −20° C. until analysis.

Individual fecal samples were taken aseptically by rectal extractionusing sterile shoulder length gloves and placed in sterile whirl packbags. Fecal samples were taken from all steers at three day intervals(3, 6, 9, 12, 15 and 18) (Table 22).

Ten grams of feces from each sample was placed into 90 ml of BismuthSulfate Broth (BSG) (Difco). Samples were mixed thoroughly and seriallydiluted ten fold (10⁻² to 10⁻⁶) using BSG as diluent. Samples wereincubated at 37 C for 24 hours. The end point for each sample wasenumerated by plating in duplicate each dilution on Brilliant GreenSalmonella selective agar. Positive cultures were confirmed byagglutination using Salmonella O antiserum (poly A-I and Vi). Thehighest dilution that had a positive Salmonella culture was determinedas the end point.

TABLE 22 Schedule of Events Daily Schedule Description of Events Day(−20) Calves were purchased from a local sales barn and upon to day (−1)entering trial facility were treated as necessary for respiratory orenteric disease, and treated for internal and external parasites. Day 0Calves were ear-tagged, randomized, and assigned to group (1st vacci- Aor B. Each calf received an initial vaccination with the nation)appropriate vaccine (test and/or placebo) subcutaneously in the neck.Serum and fecal samples were collected. Day 1, 7, 14 Monitored foradverse reactions to vaccination (local and systemic). Day 21Appropriate booster vaccination (test or placebo) was given (2^(nd)Vacci- to each calf (second vaccination). Serum samples collected.nation) Day 22, 28, Monitored for adverse reactions to vaccination(local and 37 systemic). Day 37 All groups were orally challenged with10¹² CFU of S. enterica serovar Newport challenge organism. Calvesweighed and serum samples collected. Days 38-55 Animals were monitoreddaily for morbidity and mortality. Dead calves were examined post-mortemand internal organs cultured for salmonella to confirm cause of death.Feces from all surviving calves were sampled every 3 days and culturedfor recovery of Salmonella. Day 56 Termination of trial. Remainingcalves were weighed, serum collected, and treated if necessary. Group Bcalves were vaccinated with S. enterica serovar Newport bacterialvaccine. All surviving calves were returned to a normal cattleproduction environment.

Example 12 Preparation of S. enterica Serovar Newport Challenge Organism

A frozen culture of S. enterica serovar Newport challenge organism(MS020508) was streaked on a Blood agar plate and incubated at 37° C.Several isolated colonies were transferred onto 100 ml of Tryptic SoyBroth (TSB) containing 25 μg/ml 2, 2′ dipyridyl and incubated on andorbital shaker at 200 rpm/min for 12 hours. Twenty five milliliters ofthis culture was transferred into 3500 ml of pre-warmed TSB. The culturewas incubated at 37° C. for 5 hours at which point the cells werecollected by centrifugation at 10,000×g for 20 minutes. The bacterialpellet was washed twice by centrifugation with the final pelletresuspended into 3000 ml of sterile physiological saline (0.85%). Justprior to challenge, 1 ml of the above bacterial suspension was seriallydiluted ten fold to enumerate the number of CFU per ml.

Example 13 S. enterica Serovar Newport Challenge in Holstein Calves

Sixteen days after the second vaccination all calves were orally lavagedwith 100 ml of the above bacterial suspension containing 1.0×10¹² ColonyForming Units (CFU) of S. enterica serovar Newport. Calves weremonitored daily for signs of morbidity and rectal temperatures for 18days post-challenge. Rectal temperatures were taken at three dayintervals. Fecal samples were collected approximately every 3 days forthe isolation of Salmonella. Rectal temperature and the quantitativeenumeration of Salmonella was analyzed using linear regression, whilethe likelihood of being culture-positive (yes/no) was analyzed usinglogistic regression.

Example 14 Enzyme-Linked Immunosorbent Assay (ELISA)

An Enzyme-Linked Immunosorbent Assay (ELISA) was used to monitor theserological response to vaccination. Three highly conserved proteinspresent in the composition prepared as described in Example 1 were used.Briefly, the proteins migrating at 82 kDa, 80 kDa, and 74 kDa were cutfrom unstained polyacrylamide gels. The location of these proteins wasdetermined using a stained indicator lane which was cut away from theoriginal gel and stained. Elution of the protein from the macerated gelwas carried out according to the manufacturer's recommendation using amodel 422 electro-eluter (Bio-Rad, Laboratories, Hercules, Calif.).These proteins were then used as the capture molecule in an indirectELISA test. A polyclonal antiserum was raised against the compositionderived from S. enterica serovar Newport as described in Example 1 andused as the ELISA positive control serum.

The optimum working concentrations of the purified protein andchromogenic conjugate was determined by several checkerboard titrationsusing the positive and negative control dialysates. A prediction curvewas then established to calculate protein-ELISA titers at a 1:500dilution. All subsequent tests were performed at a single serum dilution(1:500) and protein titers were calculated as an average of the testabsorbance values for each treatment.

The ELISA was performed by adding 100 μl of diluted protein in 0.05Mcarbonate buffer (pH 9.6) to each well of a 96-well flat bottom plate(Immulon 2, Dynex Technologies). After overnight incubation at 4° C.,excess protein was removed and the plate was washed. All subsequentwashing steps were done three times in phosphate buffered saline (pH7.4) with 0.05% TWEEN-20. The plates were blocked for one hour at 37° C.with 4% fish gelatin (Sigma) in PBS and then washed. Serum samples weretested in parallel at single-point dilutions (1:500) using 100 μl/welland incubated for 45 minutes at 37° C. The first two columns of eachplate contained the negative and positive control samples while the restof the plate was used for the test samples. The plate was incubated for45 minutes at 37° C. while stirring at 200 rpm. After washing, 100 μl ofa Monoclonal Anti-bovine IgG clone BG-18 Alkaline phosphatase conjugate(Sigma Chemical) at a 1:15,000 dilution was added to each well. Afterincubation for 45 minutes at 37° C., the plates were washed and 100 μlPnPP substrate (Sigma Chemical), prepared in 0.1 M glycine buffer, wasadded to each well. The substrate was allowed to react for 45 minutes at37° C. while stirring at 100 rpm. The reaction was terminated by theaddition of 25 ul of 3.0 N NaOH. The absorbance was read at 405 nm.

Example 15 Results

Calves vaccinated with the composition derived from S. enterica serovarNewport showed an enhanced serological response to vaccination whichincreased after the second vaccination showing an anamnestic responseafter the second vaccination (FIG. 4). In contrast, the placebonon-vaccinated control calves showed no antibody response.

There was a significant difference between the rectal temperature of thevaccinates (Table 23) compared to the non-vaccinated calves (Placebo)during the post challenge period (Table 24). Averaged across the studyperiod the rectal temperatures for the non-vaccinates was approximately0.4 F (95% CI=0.01-0.79 F) higher when compared to vaccinates (P=0.045).

TABLE 23 The Rectal Temperature of Vaccinated Calved following an OralChallenge with Salmonella enterica serovar Newport Calf Days PostChallenge # 0 3 4 5 6 9 12 14 18 2 103 105.1 104.8 103.7 103.7 103.7103.1 103.5 102.4 3 103.1 103.9 104.2 103.9 103.5 103.7 103.7 103.3102.4 5 103.3 105.1 103.7 105.5 103.5 104.2 103 03.1 102.4 6 105.1 104.2103.9 104.9 102.4 103.3 103 102.4 102.2 8 103.1 104.6 103.3 104.6 102.6103.7 103.3 102.5 101.9 9 103.3 104.2 104.2 103 103.1 104.6 103 102.2102.8 11 102.8 106.4 103.3 103.3 101.5 103 103.3 103 101.5 12 103.3105.8 105.1 103.9 102.6 103 102.8 102.2 101.9 14 103.7 105.1 103 103.5103 105.1 104.8 103.3 103.3 15 105.5 104.2 103.7 103.5 102.4 103 102.4102.6 102.4 17 103.7 105.1 103.9 104.2 102.4 103.3 103.3 102.4 101.3 18103.5 106.2 104.9 105.1 101.7 103 103.5 103.3 103.3 20 103.5 106.6 105.3106.2 102.8 103.3 104.2 103.3 103.7 21 103.3 104.8. 103.3 102.2 103 104103.3 103 101.3 23 N/S 105.5 105.8 103.3 102.4 103.7 103.1 102.4 102.124 N/S 105.1 105.5 104.4 103.3 103.3 103.3 102.4 102.2 26 102.8 103.9103.5 103.5 105.1 103.7 103.3 102.4 101.4 27 103 104.2 103.3 103.7 103.3103.3 103.3 103.7 102.4 29 103.3 105.5 104 105.5 105.5 106 104.6 103103.3 Mean 103.5 105.0 104.1 104.1 103.0 103.8 103.4 103 103.3Cumulative Mean = 103.6 Standard Deviation = 0.64

TABLE 24 The Rectal Temperature of Placebo-Vaccinated Calves followingan Oral Challenge with Salmonella enterica serovar Newport Days PostChallenge Calf # 0 3 4 5 6 9 12 14 18 1 103.7 106.9 105.1 105.1 103.3104.4 103.3 102.6 102.2 4 104.2 106.6 105.7 104.9 103.9 104.8 104.4103.3 102.4 7 103.7 106.4 105.7 103.3 102.8 103.7 104.2 103.3 103.9 10103.1 104.9 103.3 104.2 103 103.7 102.2 102.4 101.9 13 103.3 106.6 105.5104.2 103 105.1 104.2 102.1 103.3 16 103.7 106 104 103 102.2 103.7 102.6102.2 102.1 22 103.5 107.5 106.7 105.7 104.8 105.1 103.3 103.3 104.8 25102.8 106.9 102.8 103.3 102.4 104.9 103.5 103.3 103.3 28 104.8 105.5104.2 103.9 103.1 104.2 N/A N/A N/A Mean 103.5 106.5 104.9 104.1 103.2104.4 103.5 102.8 103 Cumulative Mean = 104.0 Standard Deviation = 1.16There was a significant difference in the amount of Salmonella beingshed between the vaccinated group compared to the non-vaccinated Placebogroup after challenge (FIG. 5). Averaged across the study period andaccounting for repeated estimates, vaccinates shed less Salmonella pergram of feces as compared to non-vaccinates (average log₁₀=0.91, 95% CIlog₁₀=0.17-64, P=0.04). Overall, among vaccinates there were about twiceas many culture-negative days. About 40% of cultured days were negativefor vaccinates (55/162) compared to non-vaccinates with 21% of culturesdays were negative (10/48). These data show that the odds of beingculture-positive for Salmonella among non-vaccinates was approximately2.5 times greater when compared to the vaccinated group (OR=2.5, 95%CI=1.24-4.93, P=0.02). These data illustrate the proof of concept that acomposition administered through vaccination prevented the colonizationof Salmonella in calves after experimental challenge.

Example 16 Decreased Fecal Shedding of E. coli O157:H7 ThroughVaccination in Holstein Steers

The purpose of this study was to evaluate the efficacy of thecomposition derived from E. coli O157:H7 in eliminating the fecalshedding of a homologous oral challenge of E. coli O157:H7 in Holsteinsteers. The immunizing composition was prepared from E. coli O157:H7 asdescribed in Examples 1 and 2. The experimental trial was initiated instarter calves on a commercial feed lot. The feed lot consisted of 500Holstein steers separated into separate grow out facilities based on theage and weight of the steers. Twelve steers (N=12) with an averageweight of approximately 300 pounds were randomly selected anddistributed into a single pen. Steers were ear tagged for identificationand randomly allocated into three groups designated as groups 1-3.Steers in Group 1 were designated as non-vaccinated and remained as thecontrol group. Steers in groups 1 and 2 were given two different vaccineformulations prepared in using two different adjuvant formulations.Steers in Group 2 were vaccinated with the vaccine adjuvanted withEMULSIGEN as described previously in Example 2, while steers in Group 3were vaccinated with the vaccine prepared in aluminum hydroxide(Rehydagel-HPA, Reheis, N.J.). Briefly, the composition was suspended in0.02M phosphate buffered saline pH 7.2 and absorbed onto aluminumhydroxide (25% vol/vol) to provide a total dose of 1000 μg in a 2 mlinjectable volume. Steers were vaccinated subcutaneously 3 times at 21day intervals. The outcome parameters used to evaluate vaccine efficacywere frequency and concentration of fecal shedding of the challengeorganism, serological response to vaccination, and injection sitereactions.

Example 17 Blood and Fecal Sample Collection

Blood samples were collected from all test steers on the initial day ofimmunization (day-0) and again at 7, 14, 21, 28, 35, 42, and 54 daysafter the first vaccination to monitor the serological response tovaccination. An Enzyme-Linked Immunosorbent Assay (ELISA) monitored theserological response to vaccination as described in Example 14 with thefollowing modification: metal regulated proteins derived from E. coliO157:H7 having molecular weights of 89 kDa, 85 kDa, 81 kDa, 78 kDa, wereused as the capture molecule in the assay. All blood was collected insterile 13×75 millimeter vacutainer collection tubes (SST No. 369783,Becton Dickinson, Franklin Lakes, N.J.). After clotting, the blood tubeswere centrifuged at 800×g for thirty minutes and frozen at −20° C.

Individual fecal samples were taken aseptically by rectal extractionusing sterile shoulder length gloves and placed in sterile whirl packbags. Fecal samples were taken from all steers on the initial day ofchallenge and again at 12, 24, 48, 72, 96, 120, 144 and 168 hours postchallenge. Briefly, ten grams of feces from each sample was placed into90 ml of physiological saline (0.85%). Samples were mixed thoroughly andserially diluted 10-fold in saline. Each dilution was plated induplicate on Eosin Methylene Blue Agar (EMB) agar containing 150 μgnalidixic acid/ml incubated at 37° C. for 48 hours. The number ofbacteria was enumerated for each sample and the log₁₀ colony formingunits was averaged for each treatment group at each time period.

Seven days after the third vaccination steers were transported from thecommercial feedlot to a Biosafety level 2 isolation facility. Steerswere equally divided among three isolation rooms so that each room hadat least one treatment group. Four days after arriving at the isolationfacility all steers were challenged. Twelve hours prior to challengefeed and water was removed from each isolation room. The E. coli O157:H7isolate as described in Example 1 was used for challenge.

Example 18 Preparation of E. coli O157:H7 Challenge Organism

To enhance the isolation rate of the E. coli O157:H7 challenge organismfrom fecal samples the nalidixic acid resistant isolate as described inExample 8 was used for challenge. Forty eight hours before challenge theisolate was removed from a frozen stock and sub-cultured onto an EMBagar plate containing 150 μg/ml nalidixic acid and incubated at 37° C.for 18 hours. A single colony was subcultured into 100 ml Tryptic SoyBroth (Difco) containing 25 μg/ml 2, 2′ dipyridyl and 150 μg nalidixicacid. The culture was incubated at 37° C. for 12 hours while rotating at200 rpm, at which point was subcultured into 4 liters Tryptic Soy Broth(Difco) containing 25 μg/ml 2, 2′ dipyridyl and 150 μg nalidixic acidincubated at 37° C. for 6 hours while continuously stirring. At the endof the incubation period the culture was centrifuged at 10,000×g for 20minutes at 4° C. to pellet the bacteria. The final bacterial pellet wasresuspended in 3600 ml of phosphate buffered saline containing 250 μg/mlnalidixic acid. Just prior to challenge, 1 ml of the above bacterialsuspension was serially diluted ten fold to enumerate the number ofCFU/calf dose.

Example 19 E. coli O157:H7 Challenge in Holstein Calves

All steers were orally lavaged with 100 ml of the above bacterialsuspension containing 4.5×10⁹ CFU. To enumerate the difference in fecalshedding between the control and vaccinated steers, individual fecalsamples were collected aseptically by rectal extraction at the time ofchallenge and again at 12, 24, 48, 72, 96, 120, 144 and 168 hours postchallenge. Blood samples were taken at the time of challenge and againat termination of the trial (168 hours).

Table 25 shows the difference in the fecal shedding between thenon-vaccinated controls (Group 1) and the vaccinated steers (Groups 2and 3). There was a highly statistical difference in the amount of E.coli O157:H7 being shed between the steers in Group 2 as compared to thenon-vaccinated/challenged controls. Averaged across the study period,steers in Group 2 shed less E. coli per gram of feces as compared to thenon-vaccinated steers of Group 1, with a degree of significance ofP=0.02 FIG. 6.

TABLE 25 The Difference in Shedding of E. coli O157:H7 Between the Non-Vaccinated and Vaccinated Treatment Groups after Challenge Mean log₁₀Colony Forming Units Group 1 Group 2 Group 3 Sampling Times(Non-Vaccinated) (Vaccinated)¹ (Vaccinated)² Challenge (Time 0) 9.7 9.79.7 12 hours 5.89 4.84 5.7 24 hours 5.51 5.3 4.77 48 hours 6.03 3.924.44 72 hours 6.12 2.1 5.4 96 hours 4.68 2.94 5 120 hours  3.72 2.3 5.4144 hours  5.55 1.4 4.4 168 hours  4.36 1.4 4.03 ¹Steers in treatmentGroup 2 were vaccinated with the composition formulated in the EMULSIGENadjuvant (22.5% vol/vol). ²Steers in treatment Group 3 were vaccinatedwith the composition formulated in aluminum hydroxide (25% vol/vol).

There was no statistical difference in the fecal shedding between thenon-vaccinated controls as compared to the shedding incidence of steersin Group 3 given the vaccine prepared in aluminum hydroxide (FIG. 7).This difference could possibly be explained due to the differentadjuvants. It is well known that oil based adjuvants often provide amuch better immune response than aluminum hydroxide based adjuvants andcould simply be due to a difference in antibody response. Nevertheless,these data illustrate for the first time that a composition as describedherein administered as a vaccine composition can prevent thecolonization of E. coli O157:H7 in calves after experimental challengegiven orally.

Example 20 Preparation of the Immunizing Compositions Derived fromSalmonella enterica Serovar Enteritidis

Metal regulated proteins were prepared from S. enterica serovarEnteritidis using the methods described in Example 1. The bacterialisolate used in this experimental study originated from a natural fieldoutbreak in a commercial chicken layer flock. Identity of the isolatewas confirmed by the Minnesota Poultry Testing Laboratory located inWillmar, Minn. and designated as MS010531. The composition prepared fromthis isolate (S. enterica serovar Enteritidis) contained the proteinsdescribed in Table 3. Two stock vaccines were prepared that representedstandard adjuvant formulations used in the poultry industry; awater-in-oil emulsion and an aqueous aluminum hydroxide formulation. Thewater-in-oil formulation was prepared by suspending the proteinsuspension in physiological saline (0.85%) containing 0.1% formalin. Theprotein concentration was standardized to contain 100 ug of protein perbird dose. Briefly, the aqueous protein suspension 250 ml was emulsifiedin a water-in-mineral oil adjuvant containing 50% DRAKEOL 6 mineral oil(Univar USA, St. Paul Minn.), 44.5% aqueous protein suspension, 2.56%TWEEN 85 (Ruger Chemical Co, Irvington, N.J.) and 3.0% SPAN 85 (RugerChemical Co, Irvington, N.J.). The mixture was emulsified using anUltra-Turrax T-50 homogenzing vessel (IKA, Cincinnati, Ohio). Thewater-in-oil emulsion was stored at 4° C.

The aqueous aluminum hydroxide formulation was prepared by suspendingthe S. Enteritidis antigen in 0.02M phosphate buffered saline pH 7.2 toa final volume of 250 ml containing 25% vol/vol aluminum hydroxide(REHYDRAGEL-HPA, Reheis, NJ) to give a final protein concentration of200 ug protein/ml.

Example 21 Chicken Vaccination and Challenge Study

Evaluation of the efficacy of the S. enterica serovar Enteritidisvaccines was carried out against a live virulent challenge in SpecificPathogen Free Chickens (SPF). Three hundred and fifty (N=350) 1-day oldSPF chicks were obtained from Charles River Spafas Inc (Roanoke, Ill.).Chicks were randomly assigned to three treatment groups, designated A,B, and C, with 116 birds in each group. Birds in Group A received a 0.1ml dose, delivered subcutaneously, of the aluminum hydroxide adjuvantvaccine at day one, followed by a 0.5 cc booster at 10 weeks of age (day70). Birds in Group B received a 0.5 cc dose, delivered subcutaneously,of the oil emulsified vaccine at 6 weeks of age (Day 42), followed by a0.5 cc booster vaccination at 10 weeks of age (Day 70). Group C servedas non-vaccinated controls.

Example 22 Intravenous Challenge with S. enteritidis Serovar Enteritidis

To enhance the isolation rate of the S. enterica serovar Enteritidischallenge organism from challenged birds the isolate was made nalidixicacid resistant as described in example 8 with the followingmodification. The S. enterica serovar Enteritidis culture was plated onBrilliant Green Agar (BG) plates rather then Eosin Methylene Blue (EMB).The nalidixic acid resistant S. enterica serovar Enteritidis isolate asprepared by the method described above was stored at −90° C. until usedfor challenge. Briefly, the isolate from a frozen stock was streakedonto a blood agar plate and incubated at 37° C. for 18 hours. A singlecolony was subcultured into 100 ml Tryptic Soy Broth (Difco) containing25 μg/ml 2, 2′ dipyridyl. The culture was incubated at 37° C. for 12hours while rotating at 200 rpm, at which point 10 ml was sub-culturedinto 500 ml pre-warmed Tryptic Soy Broth containing 25 μg/ml 2, 2′dipyridyl. The culture was incubated at 37° C. for 6 hours whilerotating at 200 rpm at which point it was centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellet was washedtwice by centrifugation in physiological saline at 4° C. The finalpellet was resuspended and aliquated into two 200 ml samples inphysiological saline. Each sample was adjusted to give a low and highchallenge dose, i.e. low dose was adjusted to 3.45×10⁷ CFU/ml while thehigh dose was adjusted to give 3.45×10⁹ CFU/ml.

Example 23 Challenge

At 12 weeks of age (day 84), all groups (A, B, and C) were separatedinto two sub-groups based on the challenge dose given, i.e. Group A wasdesignated as A₁ and A₂ while birds in Groups B and C were designated asB₁, B₂, C₁ and C₂ respectively. All birds in groups A₁, B₁, and C₁ (60birds/group) were intravenously challenged using the low dose of S.enterica serovar Enteritidis. Each bird was given 1 ml containing3.45×10⁷ CFU by intravenous injection (refer to table 26). The outcomeparameters used to evaluate vaccine efficacy in these groups was basedon the differences in the quantitative clearance of the challengedorganism from internal organs (spleen and ovaries) and difference infecal shedding as examined by culturing the cecal junction. Birds inGroups A₂, B₂, and C₂ (40 birds/group) were intravenously challengedwith the high dose of S. enterica serovar Enteritidis (3.45×10⁹ CFU/ml)to evaluate the difference in mortality between vaccinated andnon-vaccinated treatment groups. Mortality was recorded daily for 7 days(table 26).

TABLE 26 Treatment Groups, Adjuvants, and Antigen Doses # of Birds:Treatment Low High Adjuvant Antigen Priming Booster Administration Groupchlg. chlg. Formulation Treatments Dose Dose Day Group A 60 + 40 Al—OH 20 and 100 μg 0.1 ml 0.5 ml Days 1, 70 Group B 60 + 40 Oil Emulsion 100and 100 μg 0.5 ml 0.5 ml Days 42, 70 Group C 60 + 40 non-vac non-vac — ——

To enumerate differences in systemic clearance of the challenge microbefrom internal organs and intestinal colonization between vaccinates andnon-vaccinates challenged with the low dose, ten birds/group wereeuthanized by CO₂ at 12, 24 and 48 hours after challenge and the spleen,left ovary and cecal junction were aseptically removed from each bird.Each sample was individually weighted and adjusted to give a 1:10dilution (wt/vol) in physiological saline. Each sample was macerated andserially diluted ten fold. Each diluted sample was plated in duplicateon BG agar plates containing 150 ug nalidixic acid. The number ofbacteria was enumerated for each sample and the log₁₀ CFU was averagedfor each treatment group at each time period.

FIG. 8 shows the quantitative clearance of S. enterica serovarEnteritidis in spleens of vaccinated and non-vaccinated chickens, 12, 24and 48 hours after challenge. The results show a steady decline in thenumber of CFU of S. enterica serovar Enteritidis in the vaccinatedgroups for each treatment group that was statistically significant ateach sampling period as compared to the non-vaccinated controls. Thealuminum hydroxide vaccinated birds of group A₁ compared to thenon-vaccinated controls (C₁) showed a highly statistical difference ateach sampling period (12, 24 and 48 hours post challenge) with degreesof significance of P=0.036, 0.0003 and 0.024 respectively. A similarscenario was observed in birds vaccinated with the oil-emulsifiedvaccine (group B₁) showing statistically significant differences at 24and 48 hours after challenge have degrees of significance of P=0.00072and 0.0123, respectively, when compared to the non-vaccinated controls(group C₁). There was no significant difference at the 12 hour samplingperiod due to a large variation in plate counts at this time period.

FIG. 9 shows the difference in the number of CFU of S. enterica serovarEnteritidis in ovaries between vaccinated and non-vaccinated birds amongthe different treatment groups. The aluminum hydroxide vaccinated birdsof group A₁ showed statistical differences at the 24 and 48 hoursampling periods as compared to the non-vaccinated birds of group C₁with degrees of significance of P=0.005 and 0.04 respectively. The oilemulsified vaccinated birds of group B₁ also showed degrees ofsignificance at the 24 and 48 hour sampling periods with degrees ofsignificance of P=0.0048 and 0.045. As before, there was no significantdifference at the 12 hour sampling period due to variation in platecounts.

FIG. 10 shows the colonization differences or the fecal shedding of S.enterica serovar Enteritidis in the cecal junction between vaccinatedand non-vaccinated control birds. Statistical differences were seen inboth treatment groups (A₁ and B₁) when compared to non-vaccinatedcontrols at the 48 hour only sampling period with degrees ofsignificance of P=0.01 and 0.0096 respectively.

Birds in Groups A₂, B₂ and C₂ (40 birds/group) were intravenouslychallenged with the high dose of S. enterica serovar Enteritidis(3.45×10⁹ CFU/ml) to evaluate the difference in mortality betweenvaccinated and non-vaccinated treatment groups (table 27). Mortality wasrecorded at 12 hour intervals for a period of seven 7 days. There was asignificant difference in the observed mortality between birds given thealuminum hydroxide (A₂) versus the oil-emulsified adjuvanted vaccine(B₂) (Table 27). Total 7 day mortality in the aluminum hydroxide (A₂)group was 45% compared to 23% in the oil-emulsified groups (B₂) and 95%in the non-vaccinated controls of group C₂ (FIG. 94). The degree ofsignificance of group A₂ compared to the non-vaccinated control group C₂was (p=5.67×10⁻⁷) while the degree of significance of group B₂ comparedto C₂ was (p=6.79×10⁻¹²).

TABLE 27 The cumulative mortality between vaccinated treatment groupscompared to non-vaccinated controls following intravenous challenge withS. enterica serovar Enteritidis Aluminum Controls Oil Emulsion HydroxideTime Post Challenge (C2) (B2) (A2) 12 hour 0 0 0 24 hours 14  0 0 36hours 3 0 0 48 hours 5 0 0 60 hours 2 0 0 72 hours 7 0 1 84 hours 4 0 296 hours 1 1 1 108 hours 1 0 5 120 hours 1 1 3 132 hours 0 2 1 144 hours0 1 3 156 hours 0 3 1 168 hours 0 1 1 Total mortality 38/40 9/40 18/40(95%) (23%) (45%)

The results of this study demonstrate that a vaccine including proteinsisolated from S. enterica serovar Enteritidis grown under iron-limitingconditions is protective against subsequent challenge by the pathogen inlayer chickens. Birds immunized with the vaccine prepared in twocommonly used adjuvants showed a significant reduction in the number ofcolony forming units of the challenge organism (S. enterica serovarEnteritidis) from internal organs (spleen and ovaries) followingintravenous challenge compared to the non-vaccinated controls. Inaddition, vaccination also reduced the fecal shedding or colonization ofS. enterica serovar Enteritidis after challenge as compared to thenon-vaccinated controls. Both vaccination regiments used in this studyresulted in good protection against an intravenous challenge inchickens. In addition, there was minimal adverse reaction at the site ofinjection, which is a major advantage of the compositions describedherein when compared to commercially available bacterins.

Example 24 Characterization of Metal Regulated Proteins of an S.enterica Serovar Newport Isolate

The proteins of the composition prepared as described in Example 1 fromthe S. enterica serovar Newport strain were characterized using matrixassisted laser desorption/ionization time-of-flight spectrometry(MALDI-TOF MS). A portion of the composition was resolved using a sodiumdodecyl sulfate-polyacrylamide gel. After the proteins of a compositionhad been resolved, the gel stained with either coomasie brilliant blueor silver to visualize the proteins. This method was also used tocharacterize compositions obtained from S. enterica serovar Enteritidisstrain MS010531, S. enterica serovar Typhimurium strain MS010427, and S.enterica serovar IRP SDC Serial.

Materials and Methods

Excision and Washing.

The gel was washed for 10 minutes with water twice. Each protein band ofinterest was excised by cutting as close to the protein band as possibleto reduce the amount of gel present in the sample. Fourteen gelfragments were prepared, and included polypeptides having the followingapproximate molecular weights (in kilodaltons): 82 and 79 (excisedtogether in a single gel slice), 74, 65, 56, 55, 52, 45, 38 and 38(excised together in a single gel slice), 36, 22, 18, and 12.

Each gel slice was cut into 1×1 mm cubes and placed in 1.5 ml tube. Thegel pieces were washed with water for 15 minutes. All the solventvolumes used in the wash steps were approximately equal to twice thevolume of the gel slice. The gel slice was next washed withwater/acetonitrile (1:1) for 15 minutes. When the proteins had beenstained with silver, the water/acetonitrile mixture was removed, the gelpieces dried in a SpeedVac (ThermoSavant, Holbrook, N.Y.) and thenreduced and alkylated as described below. When the gel pieces were notsilver-stained, the water/acetonitrile mixture was removed, andacetonitrile was added to cover until the gel pieces turned a stickywhite, at which time the acetonitrile was removed. The gel pieces wererehydrated in 100 mM NH₄HCO₃, and after 5 minutes, a volume ofacetonitrile equal to twice the volume of the gel pieces was added. Thiswas incubated for 15 minutes, the liquid removed, and the gel piecesdried in a SpeedVac.

Reduction & Alkylation.

The dried gel pieces were rehydrated in 10 mM DTT and 100 mM NH₄HCO₃,and incubated for 45 minutes at 56° C. After allowing the tubes to coolto room temperature, the liquid was removed and the same volume of amixture of 55 mM iodoacetamide and 100 mM NH₄HCO₃ was immediately added.This was incubated for 30 minutes at room temperature in the dark. Theliquid was removed, acetonitrile was added to cover until the gel piecesturned a sticky white, at which time the acetonitrile was removed. Thegel pieces were rehydrated in 100 mM NH₄HCO₃, and after 5 minutes, avolume of acetonitrile equal to twice the volume of the gel pieces wasadded. This was incubated for 15 minutes, the liquid removed, and thegel pieces dried in a Speed vac. If the gel was stained with coomasieblue, and residual coomassie still remained, the wash with 100 mMNH₄HCO₃/acetonitrile was repeated.

In-Gel Digestion.

Gel pieces were completely dried down in a Speed Vac. The pieces wererehydrated in digestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂, 12.5nanograms per microliter (ng/μl) trypsin) at 4° C. Enough buffer wasadded to cover the gel pieces, and more was added as needed. The gelpieces were incubated on ice for 45 minutes, and the supernatant removedand replaced with 5-2 μl of same buffer without trypsin. This wasincubated at 37° C. overnight in an air incubator.

Extraction of Peptides.

A sufficient volume of 25 mM NH₄HCO₃ was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (in a bathsonicator if possible), and the supernatant was recovered. Theextraction was repeated twice, using 5% formic acid instead of NH₄HCO₃.A sufficient volume of 5% formic acid was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (typicallyin a bath sonicator), and the supernatant was recovered. The extractswere pooled, and 10 mM DTT was added to a final concentration of 1 mMDTT. The sample was dried in a SpeedVac to a final volume ofapproximately 5 μl.

Desalting of Peptides.

The samples were desalted using a ZIPTIP pipette tips (C18, Millipore,Billerica, Mass.) as suggested by the manufacturer. Briefly, a samplewas reconstituted in reconstitution solution (5:95 acetonitrile:H₂O,0.1%-0.5% trifluoroacetic acid), centrifuged, and the pH checked toverify that it was less than 3. A ZIPTIP was hydrated by aspirating 10μl of solution 1 (50:50 acetonitrile:H₂O, 0.1% trifluoroacetic acid) anddiscarding the aspirated aliquots. This was followed by aspirating 10 μlof solution 2 (0.1% trifluoroacetic acid in deionized H₂O) anddiscarding the aspirated aliquots. The sample was loaded into the tip byaspirating 10 μl of the sample slowly into the tip, expelling it intothe sample tube, and repeating this 5 to 6 times. Ten microliters ofsolution 2 was aspirated into the tip, the solution discarded byexpelling, and this process was repeated 5-7 times to wash. The peptideswere eluted by aspirating 2.5 μl of ice cold solution 3 (60:40,acetonitrile:H₂O, 0.1% trofluoroacetic acid), expelling, and thenre-aspirating the same aliquot in and out of the tip 3 times. After thesolution has been expelled from the tip, the tube is capped and storedon ice.

Mass Spectrometric Peptide Mapping.

The peptides were suspended in 10 μl to 30 μl of 5% formic acid, andanalyzed by MALDI-TOF MS (Bruker Daltonics Inc., Billerica, Mass.). Themass spectrum of the peptide fragments was determined as suggested bythe manufacturer. Briefly, a sample containing the peptides resultingfrom a tryptic digest were mixed with matrix cyano-4-hydroxycinnamicacid, transferred to a target, and allowed to dry. The dried sample wasplaced in the mass spectrometer, irradiated, and the time of flight ofeach ion detected and used to determine a peptide mass fingerprint foreach protein present in the composition. Known polypeptides were used tostandardize the machine.

Data Analysis.

The experimentally observed masses for the peptides in each massspectrum were compared to the expected masses of proteins using thePeptide Mass Fingerprint search method of the Mascot search engine(Matrix Science Ltd., London, UK, and www.matrixscience.com, see Perkinset al., Electrophoresis 20, 3551-3567 (1999)). The search parametersincluded: database, NCBInr; taxonomy, bacteria (eubacteria); type ofsearch, peptide mass fingerprint; enzyme, trypsin; fixed modifications,carbamidomethyl (C) or none; variable modifications, oxidation (M),carbamidomethyl (C), the combination, or none; mass values,monoisotopic; protein mass, unrestricted; peptide mass tolerance,between ±100 ppm and ±300 ppm or 450 ppm, or ±1 Da; peptide chargestate, Mr; max missed cleavages, 0 or 1; number of queries, 25.

Results

The result of this search was a mass fingerprint for each proteinpresent in the composition (Table 28-31).

TABLE 28 Experimental data from MALDI-TOF MS analysis of S. entericaserovar Newport Approximate molecular weight m/z value of polypeptidePolypeptide in kilodaltons fragments resulting from Designation (kDa)¹trypsin digestion² Lw221 (±300 ppm) 82 629.5 644.5 772.5 831.5 873.5991.6 1083.6 1208.6 1325.8 1378.7 1464.8 1500.7 1516.7 1619.9 1634.91635.9 1728.9 1873.1 1982.1 1998.2 2014.1 2194.1 2332.3 Lw223A (±300ppm) 80 849.5 919.5 1041.7 1098.7 1310.7 1336.8 1342.7 1365.7 1529.81565.9 1737.1 1752.0 1756.1 1847.2 1913.2 1930.2 1936.3 2032.3 2417.52588.6 2702.9 2910.8 2945.0 Lw223B (±300 ppm) 74 606.5 617.5 809.51064.5 1159.6 1211.6 1315.7 1330.8 1346.7 1528.0 1651.1 1679.0 1742.11745.9 1752.0 1794.1 1816.1 1908.2 1936.3 1954.2 1988.2 2243.4 2539.62588.6 2711.6 P4 (±300 ppm) 65 1304.60 1399.55 1509.71 1793.82 1869.901933.90 2024.94 2087.08 2258.20 Lw224 (±300 ppm) 56 1101.6 1132.8 1255.91717.1 1758.2 1960.2 2035.4 2670.8 2805.8 2861.1 3061.1 Lw225 (±300 ppm)55 959.6 1101.6 1132.7 1144.7 1255.8 1605.0 1615.0 1623.1 1641.0 1710.01717.0 1772.2 1905.2 1960.2 2085.4 2196.4 2670.7 2805.7 3060.9 Lw226(±300 ppm) 52 788.5 800.5 802.5 828.5 914.6 1090.5 1286.7 1382.8 1550.81616.9 1663.0 1739.0 1830.0 2035.1 2185.3 2209.3 2227.2 2685.6 2749.62887.6 Lw227 (±300 ppm) 45 666.5 731.5 813.5 859.5 964.5 1151.5 1224.61412.7 1423.8 1659.9 1685.0 1781.9 1966.2 2087.3 2183.2 2297.4 3101.73315.9 Lw228A (±300 ppm) 38 719.6 868.5 1058.6 1104.5 1122.6 1297.61639.9 2219.3 2383.2 2390.2 2604.4 2717.4 2758.5 2806.6 2835.5 3066.63451.7 Lw228B (±300 ppm) 38 705.6 794.5 901.5 909.6 1106.6 1205.6 1801.91835.9 1946.1 1987.0 2248.3 2383.2 3005.7 3134.7 Lw230A (±300 ppm) 36818.5 872.6 915.5 1025.5 1083.6 1157.6 1264.6 1378.7 1381.6 1537.71640.8 2303.3 2616.3 2673.4 3423.9 Lw233 (±300 ppm) 22 1051.7 1222.71588.9 1736.0 1821.1 2738.6 2853.7 3220.9 Lw234 (±300 ppm) 18 796.41263.6 1416.6 1479.7 1626.8 1797.9 2176.0 2447.1 2495.1 Lw235 (±300 ppm)12 789.5 1246.6 1258.6 1361.7 1531.9 2061.1 2874.6 ¹Molecular weight, inkilodaltons, of polypeptide obtained from S. enterica serovar Newport.²m/z, mass (m) to charge (z) ratio. Each m/z value includes a range ofplus or minus 300 ppm.

TABLE 29 Experimental data from MALDI-TOF MS analysis of S. entericaserovar Enteritidis Approximate molecular weight m/z value ofpolypeptide Polypeptide in kilodaltons fragments resulting fromDesignation (kDa)¹ trypsin digestion² Lw98 (±1 Da) 92 729.61 816.54889.51 958.56 973.62 987.62 999.54 1009.56 1048.62 1077.64 1114.611181.55 1220.62 1277.56 1283.77 1339.65 1385.61 1402.70 1403.69 1471.701520.84 1528.74 1649.80 1692.96 1713.93 1733.89 1759.91 1787.97 1895.031955.01 2160.25 2255.09 2286.19 2794.68 2882.59 Lw99 (±1 Da) 91 905.48925.41 946.49 1005.46 1051.45 1077.49 1110.47 1200.52 1277.51 1295.471308.60 1344.52 1376.58 1418.64 1451.54 1510.49 1511.63 1602.79 1619.681625.71 1669.65 1767.85 1768.86 1793.81 1809.83 1833.85 2014.04 2089.132269.98 2299.06 2454.09 2255.19 2573.21 Lw101 (±1 Da) 86 644.6 873.5951.5 991.5 1083.6 1085.5 1096.4 1152.6 1182.4 1208.5 1325.6 1366.51378.5 1412.6 1433.6 1464.6 1500.5 1561.7 1562.7 1585.6 1619.6 1634.71728.7 1871.8 1904.9 1975.9 1981.9 1998.0 2078.9 2193.0 2234.1 2372.02532.2 2623.3 2634.1 3099.3 3212.4 3474.5 Lw102 (±1 Da) 83 611.5 629.6849.4 919.4 1041.5 1098.5 1142.4 1154.5 1163.4 1219.5 1310.4 1336.51342.5 1365.4 1406.6 1461.6 1529.5 1565.6 1736.7 1752.7 1755.7 1846.81881.9 1912.9 1954.9 2031.9 2262.0 2399.0 2417.1 2702.3 2910.4 2944.5Lw103 (±1 Da) 78 606.5 615.6 617.6 809.4 837.4 990.4 1061.4 1064.41142.4 1159.4 1178.5 1211.4 1315.5 1330.6 1346.4 1494.6 1527.6 1571.71650.8 1655.8 1741.8 1745.6 1751.8 1793.9 1815.8 1907.9 1953.9 2243.15239.4 2711.3 Lw104 (±1 Da) 55 788.5 802.5 914.6 1180.5 1227.5 1286.51382.6 1550.7 1616.8 1662.8 1738.8 1829.9 2035.0 2185.1 2209.0 2227.12749.3 2887.5 Lw106A (±1 Da) 40 692.5 705.6 901.5 909.6 974.5 1106.61129.4 1192.5 1205.5 1439.6 1801.9 1891.9 1991.0 2248.2 2340.3 2406.23005.7 Lw106B (±1 Da) 39 719.6 868.5 1058.5 1104.5 1122.5 1280.5 1297.51640.8 1891.9 2219.2 2383.2 2390.2 2758.5 2806.6 3067.6 Lw108 (±1 Da) 38818.5 1025.5 1083.6 1222.7 1233.6 1264.7 1378.8 1381.7 1470.8 1537.91641.0 2303.5 2616.7 2673.8 3424.3 ¹Molecular weight, in kilodaltons, ofpolypeptide obtained from S. enterica serovar Enteritidis. ²m/z, mass(m) to charge (z) ratio. Each m/z value includes a range of plus orminus 1 Dalton.

TABLE 30 Experimental data from MALDI-TOF MS analysis of S. entericaserovar Typhimurium Approximate molecular weight m/z value ofpolypeptide Polypeptide in kilodaltons fragments resulting fromDesignation (kDa)¹ trypsin digestion² Lw111 (±1 Da) 86 991.6 1083.71182.6 1208.6 1307.7 1325.8 1378.7 1433.8 1478.8 1500.8 1585.8 1618.91619.9 1634.9 1659.9 1729.0 1872.1 1982.1 1998.2 2022.2 2079.2 2119.42194.2 2204.2 2332.3 2374.3 2633.5 3099.9 Lw112 (±1 Da) 82 611.5 849.5919.5 1041.6 1095.6 1098.6 1154.7 1163.6 1209.6 1219.7 1310.7 1336.71342.7 1365.7 1406.8 1529.8 1565.9 1566.9 1737.0 1756.0 1847.1 1882.21884.1 1913.2 1931.2 1955.2 2032.2 2192.4 2262.3 2417.4 2449.4 2702.22910.7 2944.9 Lw113 (±1 Da) 77 958.5 1159.5 1179.5 1211.5 1309.7 1315.61330.7 1346.5 1398.7 1527.7 1650.9 1655.9 1745.7 1751.9 1793.7 1954.02022.1 2202.1 2243.1 Lw115A (±1 Da) 40 652.6 705.7 794.6 901.6 909.61106.7 1120.6 1129.5 1175.6 1205.5 1348.8 1439.8 1802.0 1835.9 1987.12248.1 2340.2 2406.1 3005.6 3134.5 Lw115B (±1 Da) 39 719.7 868.6 1058.71104.6 1122.7 1161.6 1280.7 1297.5 2219.1 2383.1 2390.1 2758.3 2806.43451.5 Lw117 (±1 Da) 38 645.6 818.5 872.6 915.6 943.5 1025.5 1043.61083.5 1141.6 1222.6 1264.6 1378.7 1381.6 1470.6 1537.7 1640.7 1709.82303.2 2616.3 2627.2 2673.4 3423.7 3540.8 ¹Molecular weight, inkilodaltons, of polypeptide obtained from S. enterica serovarTyphimurium. ²m/z, mass (m) to charge (z) ratio. Each m/z value includesa range of plus or minus 1 Dalton.

TABLE 31 Experimental data from MALDI-TOF MS analysis of S. entericaserovar Dublin. Approximate molecular weight m/z value of polypeptide inkilodaltons fragments resulting from Polypeptide Designation (kDa)¹trypsin digestion² Dublin-1 (±300 ppm) 96 1083.43 1208.36 1315.321378.40 1500.50 1516.48 1634.65 1728.66 1871.78 1956.88 1981.83 1997.932013.92 2119.03 2193.82 2203.84 2209.82 2331.9 Dublin-2 (±1 Da) 89611.32 629.38 849.27 919.23 1041.34 1098.34 1219.28 1310.27 1336.361342.31 1365.25 1529.35 1565.39 1736.52 1752.52 1846.58 1881.64 1912.692262.75 2416.81 2702.01 2910.99 Dublin-3 (±1 Da) 81 606.31 617.37 990.191064.17 1178.19 1315.21 1330.34 1527.30 1650.36 1741.45 1745.26 1751.391793.44 1815.42 1907.47 1936.49 1953.31 2196.69 2242.57 2552.77 2587.732710.65 Dublin-4 (±1 Da) 61 632.32 945.20 1101.13 1116.16 1164.151317.16 1475.24 1764.30 1833.30 2007.47 2084.57 2669.63 2683.59 2859.91Dublin-5 (±1 Da) 56 914.20 989.18 1286.12 1382.20 1550.21 1616.311662.31 1738.31 1829.32 2034.37 2185.46 2208.44 Dublin-6 (±1 Da) 51945.18 1116.13 1221.10 1317.12 1445.06 1475.17 1815.31 1833.26 2007.392669.52 2683.47 2859.77 Dublin-7 (±450 ppm) 43 1172.16 1188.14 1343.121376.05 1392.04 1423.19 1527.29 1854.31 2344.42 2360.41 3078.65 Dublin-8(±1 Da) 40 1205.09 1348.22 1439.14 1802.27 1836.26 2247.49 2339.522405.42 3004.81 Dublin-9,10,11 (±1 Da) 38 818.24 1025.36 1083.42 1264.261378.64 1381.53 1640.41 2302.91 2615.82 2672.85 3423.18 ¹Molecularweight, in kilodaltons, of polypeptide obtained from S. enterica serovarDublin. ²m/z, mass (m) to charge (z) ratio. Each m/z value includes arange of plus or minus 300 ppm (the polypeptide Dublin-1), 450 ppm (thepolypeptide Dublin-7), or 1 Dalton (the remaining polypeptides).

Example 25 Characterization of Metal Regulated Proteins of E. coli

The proteins of the composition prepared as described in Example 1 fromthe E. coli strain BEcO157(stx-) were characterized using MALDI-TOF MSas described in Example 24. Twelve gel fragments were prepared, andincluded polypeptides having the following approximate molecular weights(in kilodaltons): 90, 86, 83, 79, a doublet at 66, 56, 38, 37, and 29.These methods were also used for the the characterization of proteins ofthe E. coli strains MS040330, MS040324, and MS040827.

Results

The result of this search was a mass fingerprint for each proteinpresent in the composition (Table 32-35).

TABLE 32 Experimental data from MALDI-TOF MS analysis of E. coli strainBEcO157(stx-). Approximate molecular weight m/z value of polypeptide inkilodaltons fragments resulting from Polypeptide Designation (kDa)¹trypsin digestion² Lw118 (±1 Da) 90 629.7 772.6 831.5 991.6 1178.61285.6 1321.7 1369.7 1433.8 1516.8 1619.9 1634.9 1706.9 1788.0 1798.01872.1 1966.2 1982.1 2089.1 2175.1 2303.2 2601.5 2706.6 2844.6 3082.83213.8 Lw119 (±1 Da) 86 976.5 992.6 1095.5 1247.6 1278.7 1359.7 1396.51436.7 1493.6 1572.9 1650.8 1665.8 1810.9 1914.0 2134.3 2211.2 2270.1LW-1A-3 (±300 ppm) 83 837.27 884.29 1048.20 1127.26 1338.26 1351.291397.26 1472.33 1621.46 1650.47 1722.49 1727.41 1759.47 1813.46 1829.432282.75 2512.79 Lw121 (±1 Da) 79 716.5 952.5 1135.5 1155.7 122.6 1336.61396.6 1447.7 1512.7 1532.7 1653.8 1657.8 1677.9 1717.9 1780.0 1861.01964.1 2262.2 2398.2 LW-1A-5A (±450 ppm) 66 632.33 945.20 1191.311238.16 1440.25 1561.26 1647.24 1792.36 2062.63 2085.55 2190.41 2248.512454.66 2628.63 LW-1A-5B (±450 ppm) 66 679.45 1295.14 1299.04 1304.171423.20 1550.34 1820.33 1892.48 1918.33 2159.57 2323.52 2357.55 2699.58LW-1A-6 (±450 ppm) 56 1285.15 1395.19 1550.25 1616.35 1829.34 2034.422139.53 2183.52 2242.47 Lw123 (±1 Da) 38 705.5 885.4 931.5 939.4 1120.41122.5 1124.4 1171.4 1290.4 1348.5 1379.4 1380.5 1439.5 1664.6 1820.71821.7 2232.9 2353.9 2447.9 2585.1 2792.1 2991.3 3106.3 Lw124 (±1 Da) 37818.4 915.4 1027.5 1055.3 1083.4 1155.4 1222.5 1280.4 1378.5 1409.41443.5 1565.5 1654.6 1709.7 2231.9 2600.1 2601.0 2671.0 3478.4 LW-1A-10(±300 ppm) 29 951.32 1020.31 1485.37 1501.36 1517.50 1676.54 1692.47¹Molecular weight, in kilodaltons, of polypeptide obtained from E. colistrain BEcO157(stx-). ²m/z, mass (m) to charge (z) ratio. Each m/z valueincludes a range of plus or minus 300 ppm (the 83 kDa and 29 kDapolypeptides), 450 ppm (the 66 kDa and 56 kDa polypeptides), or 1 Dalton(the remaining polypeptide).

TABLE 33 Experimental data from MALDI-TOF MS analysis of E. coli strainMS040330. Approximate molecular weight m/z value of polypeptide inkilodaltons fragments resulting from Polypeptide Designation (kDa)¹trypsin digestion² AB1-1 (±250 ppm) 92 905.38 909.28 1051.29 1079.321172.32 1277.26 1344.32 1404.39 1467.34 1480.42 1511.37 1547.30 1568.401625.45 1641.44 1669.39 1685.37 1740.54 1823.54 1859.56 2122.66 2140.64AB1-2 (±300 ppm) 80 629.56 831.31 1178.28 1634.37 1787.37 1787.371797.30 1871.47 1981.45 2088.38 2091.59 2174.41 2303.43 2843.60 AB1-3(±400 ppm) 77 1381.12 1526.19 1689.32 1750.32 1832.33 1889.45 1929.361968.43 1984.43 2031.32 2918.52 2959.75 AB1-4 (±400 ppm) 72 629.51808.24 872.25 889.19 1739.11 1763.14 1873.25 1999.31 2104.23 2141.272207.32 2415.36 2439.43 AB1-5 (±500 ppm) 66 615.37 716.27 771.26 831.08942.06 952.10 1026.17 1150.99 1155.12 1222.04 1335.99 1395.96 1531.961657.93 1673.00 1677.03 1717.11 1779.09 1963.12 1998.11 2261.12 2397.123305.10 AB1-6 (±450 ppm) 50 788.24 802.23 828.20 914.20 1180.04 1345.961737.18 1829.11 2035.14 2184.22 2185.23 2227.19 AB1-7 (±400 ppm) 42632.41 709.30 716.31 760.25 931.16 1003.12 1020.15 2248.21 2642.262700.21 2815.45 AB1-8 (±500 ppm) 38 705.30 842.18 885.07 1289.95 1439.002553.13 2990.28 AB1-9 (±450 ppm) 36 719.37 868.16 1058.17 1249.011439.07 1934.06 2217.24 2389.19 2834.32 AB1-10 (±400 ppm) 35 818.29834.28 872.28 1055.16 1280.19 1378.26 1423.20 1640.26 2231.49 2599.583495.73 AB1-11 (±300 ppm) 30 707.41 777.48 930.38 965.44 1066.41 1082.381109.37 1205.37 1221.32 1404.52 1577.47 1592.47 2392.78 Lw214 (±1 Da) 19915.8 942.7 951.8 1020.9 1486.2 1518.3 1605.2 1677.4 1679.3 Lw215 (±1Da) 16 603.5 915.6 942.5 951.6 1020.6 1043.7 1148.7 1363.7 1485.8 1518.01604.9 1677.0 1769.0 1933.1 2263.3 ¹Molecular weight, in kilodaltons, ofpolypeptide obtained from E. coli strain MS040330. ²m/z, mass (m) tocharge (z) ratio. Each m/z value includes a range of plus or minus 250ppm (the 92 kDa polypeptide), plus or minus 300 ppm (the 80 kDa and 30kDa polypeptides), plus or minus 400 ppm (the 77 kDa, 72 kDa, 42 kDa,and 35 kDa polypeptides), plus or minus 450 ppm (the 50 kDa and 36 kDapolypeptides), plus or minus 500 ppm (the 66 kDa and 38 kDapolypeptides) or 1 Dalton (the 19 kDa and 16 kDa polypeptides).

TABLE 34 Experimental data from MALDI-TOF MS analysis of E. coli strainMS040324. Approximate molecular weight m/z value of polypeptide inkilodaltons fragments resulting from Polypeptide Designation (kDa)¹trypsin digestion² J4-1 (±400 ppm) 82 629.46 1307.21 1532.22 1579.331634.33 1787.34 1797.32 1981.42 2089.41 2091.56 2126.48 2843.61 J4-2(±300 ppm) 79 686.54 737.44 842.49 861.44 1147.48 1163.44 1208.431244.42 1279.62 1473.59 1487.60 1579.67 1616.67 1718.71 2014.87 2036.812110.97 2126.95 J4-3 (±300 ppm) 88 650.52 672.53 821.37 1124.32 1279.501297.39 1325.37 1381.37 1424.43 1551.45 1703.56 1732.51 2024.70 2036.672251.76 2787.11 2847.98 J4-4 (±300 ppm) 60 676.37 679.52 1756.36 1820.391892.53 1932.42 2024.43 2159.65 2207.56 2255.71 2323.60 2357.64 2699.70J4-5 (±400 ppm) 54 788.28 802.28 828.24 914.26 1231.12 1285.15 1323.251346.10 1550.24 1616.31 1737.36 1829.31 2034.39 2139.50 2183.47 2242.46J4-6 (±350 ppm) 46 731.33 859.24 964.18 1563.21 1579.20 1677.22 1684.351731.31 2132.45 2148.46 2210.46 2371.41 2387.42 3216.50 J4-7 (±400 ppm)45 786.26 1025.13 1030.16 1255.15 1497.14 1505.19 1587.22 1652.171794.25 1899.36 1998.32 Lw216 (±300 ppm) 38 868.5 1249.7 1439.8 1935.12218.4 2390.4 2602.6 2977.6 3308.1 Lw217 (±1 Da) 37 818.4 1280.6 1378.72232.2 2600.5 2602.4 J4-11 (±300 ppm) 31 707.35 777.40 930.33 965.351066.31 1082.29 1109.27 1205.26 1221.24 1404.42 1576.39 1576.39 1592.382392.69 J4-12 (±400 ppm) 30 717.33 1339.16 1463.13 1841.21 1857.181882.30 1898.31 2263.24 2825.38 2868.54 Lw218 (±1 Da) 19 915.6 942.5951.6 1020.6 1363.7 1485.8 1518.0 1604.9 1677.0 1679.0 1756.1 1933.22263.4 Lw219 (±1 Da) 16 603.3 915.5 942.4 951.5 1020.5 1043.6 1148.61363.5 1485.6 1517.8 1604.8 1676.8 1755.9 1768.9 1932.9 2263.1¹Molecular weight, in kilodaltons, of polypeptide obtained from E. colistrain MS040324. ²m/z, mass (m) to charge (z) ratio. Each m/z valueincludes a range of plus or minus 300 ppm (the 88 kDa, 79 kDa, 60 kDa,38 kDa, and 31 kDa polypeptides), plus or minus 350 ppm (the 46 kDapolypeptide), plus or minus 400 ppm (the 82 kDa, 54 kDa, 45 kDa, and 30kDa polypeptides), or plus or minus 1 Dalton (the 37 kDa, 19 kDa and 16kDa polypeptides).

TABLE 35 Experimental data from MALDI-TOF MS analysis of E. coli strainMS040827. Approximate molecular weight m/z value of polypeptide inkilodaltons fragments resulting from Polypeptide Designation (kDa)¹trypsin digestion² Lw189A (±300 ppm) 101 889.38 987.47 999.42 1009.401114.54 1277.56 1339.61 1402.67 1471.65 1520.80 1528.76 1699.95 1759.951771.84 1955.01 2146.22 2156.16 2239.30 2255.14 2912.67 Lw189B (±300ppm) 101 905.46 1172.53 1277.56 1295.50 1308.56 1344.62 1404.72 1451.671547.72 1669.78 1718.76 1764.93 1823.94 1833.02 1860.08 2014.15 2089.24Lw190 (±1 Da) 88 1178.44 1307.50 1517.55 1579.72 1634.75 1787.82 1797.761871.89 1981.92 2127.06 2174.98 2303.05 2707.40 2844.30 3082.43 3197.45Lw191 (±1 Da) 85 565.45 686.41 737.43 861.47 862.47 1147.52 1208.531244.54 1279.54 1280.52 1330.57 1487.72 1579.76 1616.78 1651.80 1697.791718.84 2014.99 2036.95 2111.03 2222.12 2409.18 2582.31 2684.41 2947.433022.60 3145.50 Lw193 (±1 Da) 77 524.21 650.30 821.32 1124.35 1160.351279.56 1297.44 1325.41 1373.52 1381.40 1424.49 1510.59 1551.48 1554.631650.61 1703.67 1924.87 2013.84 2024.77 2204.93 2251.87 2553.03 2786.142848.01 3195.23 3337.27 3386.31 Lw194 (±1 Da) 67 679.56 1113.58 1136.531244.65 1260.59 1295.59 1299.46 1304.58 1350.56 1423.69 1522.66 1550.841756.84 1762.81 1887.91 1893.05 1932.95 2025.00 2159.25 2207.14 2256.182324.18 Lw195 (±150 ppm) 38 1058.57 1249.50 1439.66 1934.81 1960.972218.06 2390.13 2977.49 3357.61 3550.53 Lw196 (±1 Da) 35 645.43 818.41834.41 872.52 915.47 1055.45 1083.47 1155.48 1214.51 1222.57 1249.471280.55 1370.65 1378.65 1409.55 1654.76 2062.93 2078.94 2232.10 2601.213494.61 ¹Molecular weight, in kilodaltons, of polypeptide obtained fromE. coli strain MS040827. ²m/z, mass (m) to charge (z) ratio. Each m/zvalue includes a range of plus or minus 150 ppm (the 38 kDa and 35 kDapolypeptide), plus or minus 300 ppm (the 101 kDa polypeptides), or plusor minus 1 Dalton (the 88 kDa, 85 kDa, 77 kDa, and 67 kDa polypeptides).

Example 26 Comparison of Proteins Similar to Those Expressed by E. coliwith Other Proteins

The proteins derived from E. coli and Salmonella grown underiron-limiting conditions were identified by MALDI-TOF MS. These analysesresulted in protein sequences that represent the best protein match foreach peptide mass fingerprint (see Tables 10-17). The publicavailability of genomic sequence data allows for a database search forthese proteins in other organisms. Thus, nine of the proteins (ChuA,Imp, TolC, R4, LamB, BtuB, IutA, FepA, and FecA) identified from the E.coli strains were used in BLAST searches to determine which otherpathogens may also express these proteins. Eight of the ten selectedproteins were very similar to proteins from Shigella spp., with 98 to99% identity at the amino acid level (FIG. 11). Several of the E. coliproteins were also similar to proteins from Salmonella, Yersinia,Klebsiella, and Pseudomonas spp. These analyses suggest thatcompositions derived from E. coli grown under iron-limiting conditionsmay constitute antigens that will protect against other pathogens,particularly Shigella species.

The complete disclosure of all patents, patent applications,publications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A composition comprising: a first isolatediron-regulated polypeptide from E. coli strain BEcO157(stx-), depositedunder ATCC accession number PTA-124618, having a molecular weight of 90kDa as measured following electrophoresis on an SDS-polyacrylamide gel,wherein the first isolated iron-regulated polypeptide, if digested withtrypsin, produces polypeptide fragments having m/z values of 629.7±300parts per million (ppm), 772.6±300 ppm, 831.5±300 ppm, 991.6±300 ppm,1178.6±300 ppm, 1285.6±300 ppm, 1321.7±300 ppm, 1369.7±300 ppm,1433.8±300 ppm, 1516.8±300 ppm, 1619.9±300 ppm, 1634.9±300 ppm,1706.9±300 ppm, 1788.0±300 ppm, 1798.0±300 ppm, 1872.1±300 ppm,1966.2±300 ppm, 1982.1±300 ppm, 2089.1±300 ppm, 2175.1±300 ppm,2303.2±300 ppm, 2601.5±300 ppm, 2706.6±300 ppm, 2844.6±300 ppm,3082.8±300 ppm, and 3213.8±300 ppm; a second isolated iron-regulatedpolypeptide from E. coli strain BEcO157(stx-), deposited under ATCCaccession number PTA-124618, having a molecular weight of 86 kDa asmeasured following electrophoresis on an SDS-polyacrylamide gel, whereinthe second isolated iron-regulated polypeptide, if digested withtrypsin, produces polypeptide fragments having m/z values of 976.5±300ppm, 992.6±300 ppm, 1095.5±300 ppm, 1247.6±300 ppm, 1278.7±300 ppm,1359.7±300 ppm, 1396.5±300 ppm, 1436.7±300 ppm, 1493.6±300 ppm,1572.9±300 ppm, 1650.8±300 ppm, 1665.8±300 ppm, 1810.9±300 ppm,1914.0±300 ppm, 2134.3±300 ppm, 2211.2±300 ppm, and 2270.1±300 ppm; athird isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 83 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the third isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving m/z values of 837.27±300 ppm, 884.29±300 ppm, 1048.20±300 ppm,1127.26±300 ppm, 1338.26±300 ppm, 1351.29±300 ppm, 1397.26±300 ppm,1472.33±300 ppm, 1621.46±300 ppm, 1650.47±300 ppm, 1722.49±300 ppm,1727.41±300 ppm, 1759.47±300 ppm, 1813.46±300 ppm, 1829.43±300 ppm,2282.75±300 ppm, and 2512.79±300 ppm; a fourth isolated iron-regulatedpolypeptide from E. coli strain BEcO157(stx-), deposited under ATCCaccession number PTA-124618, having a molecular weight of 79 kDa asmeasured following electrophoresis on an SDS-polyacrylamide gel, whereinthe fourth isolated iron-regulated polypeptide, if digested withtrypsin, produces polypeptide fragments having m/z values of 716.5±300ppm, 952.5±300 ppm, 1135.5±300 ppm, 1155.7±300 ppm, 1336.6±300 ppm,1396.6±300 ppm, 1447.7±300 ppm, 1512.7±300 ppm, 1532.7±300 ppm,1653.8±300 ppm, 1657.8±300 ppm, 1667.9±300 ppm, 1717.9±300 ppm,1780.0±300 ppm, 1861.0±300 ppm, 1964.1±300 ppm, 2262.2±300 ppm, and2398.2 Da±300 ppm; a fifth isolated iron-regulated polypeptide from E.coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 66 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the fifth isolatediron-regulated polypeptide, if digested with trypsin, producespolypeptide fragments having m/z values of 632.33±450 ppm, 945.20±450ppm, 1191.31±450 ppm, 1238.16±450 ppm, 1440.25±450 ppm, 1561.26±450 ppm,1647.24±450 ppm, 1792.36±450 ppm, 2062.63±450 ppm, 2085.55±450 ppm,2190.41±450 ppm, 2248.51±450 ppm, 2454.66±450 ppm, and 2628.63±450 ppm;a sixth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 66 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the sixth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving m/z values of 679.45±450 ppm, 1295.14±450 ppm, 1299.04±450 ppm,1304.17±450 ppm, 1423.20±450 ppm, 1550.34±450 ppm, 1820.33±450 ppm,1892.48±450 ppm, 1918.33±450 ppm, 2159.57±450 ppm, 2323.52±450 ppm,2357.55±450 ppm, and 2699.58±450 ppm; and an effective amount of apharmaceutically acceptable adjuvant.
 2. The composition of claim 1further comprising a seventh isolated iron-regulated polypeptide from E.coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 56 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the seventhisolated iron-regulated polypeptide, if digested with trypsin, producespolypeptide fragments having m/z values of 1285.15±450 ppm, 1395.19±450ppm, 1550.25±450 ppm, 1616.35±450 ppm, 1829.34±450 ppm, 2034.42±450 ppm,2139.53±450 ppm, 2183.52±450 ppm, and 2242.47±450 ppm; an eighthisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 38 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the eighth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving m/z values of 705.5±450 ppm, 885.4±450 ppm, 931.5±450 ppm,939.4±450 ppm, 1120.4±450 ppm, 1122.5±450 ppm, 1124.4±450 ppm,1171.4±450 ppm, 1290.4±450 ppm, 1348.5±450 ppm, 1379.4±450 ppm,1380.5±450 ppm, 1439.5±450 ppm, 1664.6±450 ppm, 1820.7±450 ppm,1821.7±450 ppm, 2232.9±450 ppm, 2353.9±450 ppm, 2447.9±450 ppm,2585.1±450 ppm, 2792.1±450 ppm, 2991.3±450 ppm, and 3106.3±450 ppm; aninth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 37 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the ninth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving m/z values of 818.4±300 ppm, 915.4±300 ppm, 1207.5±300 ppm,1055.3±300 ppm, 1083.4±300 ppm, 1155.4±300 ppm, 1222.5±300 ppm,1280.4±300 ppm, 1378.5±300 ppm, 1409.4±300 ppm, 1443.5±300 ppm,1565.5±300 ppm, 1654.6±300 ppm, 1709.7±300 ppm, 2231.9±300 ppm,2600.1±300 ppm, 2601.0±300 ppm, 2671.0±300 ppm, and 3478.4±300 ppm; or atenth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 29 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the tenth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving m/z values of 951.32±300 ppm, 1020.31±300 ppm, 1485.37±300 ppm,1501.36±300 ppm, 1517.50±300 ppm, 1676.54±300 ppm, and 1692.47±300 ppm.3. A method of inducing production in an animal of antibody thatspecifically binds an iron regulated polypeptide isolated from E. colistrain BEcO157(stx-), deposited under ATCC accession number PTA-124618,the method comprising: administering to the animal an effective amountof the composition of claim
 1. 4. The method of claim 3 wherein theanimal is avian, bovine, caprine, ovine, porcine, bisontine, cervine,equine, a companion animal, or human.
 5. The method of claim 4 whereinthe method further comprises collecting the antibody from the animal. 6.The method of claim 5 wherein the method further comprises isolating theantibody.
 7. A composition comprising: a first isolated iron-regulatedpolypeptide from E. coli strain BEcO157(stx-), deposited under ATCCaccession number PTA-124618, having a molecular weight of 90 kDa asmeasured following electrophoresis on an SDS-polyacrylamide gel, whereinthe first isolated iron-regulated polypeptide, if digested with trypsin,produces polypeptide fragments having the amino acid sequences of SEQ IDNO:716 to SEQ ID NO:741; a second isolated iron-regulated polypeptidefrom E. coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 86 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the secondisolated iron-regulated polypeptide, if digested with trypsin, producespolypeptide fragments having the amino acid sequences of SEQ ID NO:742to SEQ ID NO:758; a third isolated iron-regulated polypeptide from E.coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 83 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the third isolatediron-regulated polypeptide, if digested with trypsin, producespolypeptide fragments having the amino acid sequences of SEQ ID NO:759to SEQ ID NO:772, SEQ ID NO:774, and SEQ ID NO:775; a fourth isolatediron-regulated polypeptide from E. coli strain BEcO157(stx-), depositedunder ATCC accession number PTA-124618, having a molecular weight of 79kDa as measured following electrophoresis on an SDS-polyacrylamide gel,wherein the fourth isolated iron-regulated polypeptide, if digested withtrypsin, produces polypeptide fragments having the amino acid sequencesof SEQ ID NO:776 to SEQ ID NO:794; a fifth isolated iron-regulatedpolypeptide from E. coli strain BEcO157(stx-), deposited under ATCCaccession number PTA-124618, having a molecular weight of 66 kDa asmeasured following electrophoresis on an SDS-polyacrylamide gel, whereinthe fifth isolated iron-regulated polypeptide, if digested with trypsin,produces polypeptide fragments having the amino acid sequences of SEQ IDNO:795 to SEQ ID NO:808; a sixth isolated iron-regulated polypeptidefrom E. coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 66 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the sixth isolatediron-regulated polypeptide, if digested with trypsin, producespolypeptide fragments having the amino acid sequences of SEQ ID NO:809to SEQ ID NO:821; and an effective amount of a pharmaceuticallyacceptable adjuvant.
 8. The composition of claim 7 further comprising: aseventh isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 56 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the seventh isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving the amino acid sequences of SEQ ID NO:822 to SEQ ID NO:830; aneighth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 38 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the eighth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving the amino acid sequences of SEQ ID NO:831 to SEQ ID NO:853; aninth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 37 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the ninth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving the amino acid sequences of SEQ ID NO:854 to SEQ ID NO:872; or atenth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 29 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the tenth isolated iron-regulatedpolypeptide, if digested with trypsin, produces polypeptide fragmentshaving the amino acid sequences of SEQ ID NO:873 to SEQ ID NO:875, SEQID NO:877, and SEQ ID NO:878.
 9. A method of inducing production in ananimal of antibody that specifically binds an iron regulated polypeptideisolated from E. coli strain BEcO157(stx-), deposited under ATCCaccession number PTA-124618, the method comprising: administering to theanimal an effective amount of the composition of claim
 7. 10. The methodof claim 9 wherein the animal is avian, bovine, caprine, ovine, porcine,bisontine, cervine, equine, a companion animal, or human.
 11. The methodof claim 6 wherein the method further comprises collecting the antibodyfrom the animal.
 12. The method of claim 11 wherein the method furthercomprises isolating the antibody.
 13. A composition comprising: a firstisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 90 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the first isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:716, SEQ IDNO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQID NO:722, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727,SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ IDNO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:738, SEQID NO:739, SEQ ID NO:740, and SEQ ID NO:741, a second isolatediron-regulated polypeptide from E. coli strain BEcO157(stx-), depositedunder ATCC accession number PTA-124618, having a molecular weight of 86kDa as measured following electrophoresis on an SDS-polyacrylamide gel,wherein the second isolated iron-regulated polypeptide comprises theamino acid sequences of SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749,SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:753, SEQ ID NO:754, SEQ IDNO:755, SEQ ID NO:756, SEQ ID NO:757, and SEQ ID NO:758; a thirdisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 83 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the third isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:759, SEQ IDNO:760, SEQ ID NO:761, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:770,SEQ ID NO:771, SEQ ID NO:772, SEQ ID NO:774, and SEQ ID NO:775; a fourthisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 79 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the fourth isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:776, SEQ IDNO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:780, SEQ ID NO:781, SEQID NO:782, SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ ID NO:786,SEQ ID NO:787, SEQ ID NO:788, SEQ ID NO:789, SEQ ID NO:790, SEQ IDNO:791, SEQ ID NO:792, SEQ ID NO:793, and SEQ ID NO:794; a fifthisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 66 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the fifth isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:795, SEQ IDNO:796, SEQ ID NO:797, SEQ ID NO:798, SEQ ID NO:799, SEQ ID NO:800, SEQID NO:801, SEQ ID NO:802, SEQ ID NO:803, SEQ ID NO:804, SEQ ID NO:805,SEQ ID NO:806, SEQ ID NO:807, and SEQ ID NO:808; a sixth isolatediron-regulated polypeptide from E. coli strain BEcO157(stx-), depositedunder ATCC accession number PTA-124618, having a molecular weight of 66kDa as measured following electrophoresis on an SDS-polyacrylamide gel,wherein the sixth isolated iron-regulated polypeptide comprises theamino acid sequences of SEQ ID NO:809, SEQ ID NO:810, SEQ ID NO:811, SEQID NO:812, SEQ ID NO:814, SEQ ID NO:815, SEQ ID NO:816, SEQ ID NO:817,SEQ ID NO:818, SEQ ID NO:819, SEQ ID NO:820, and SEQ ID NO:821; and aneffective amount of a pharmaceutically acceptable adjuvant.
 14. Thecomposition of claim 13 further comprising: a seventh isolatediron-regulated polypeptide from E. coli strain BEcO157(stx-), depositedunder ATCC accession number PTA-124618, having a molecular weight of 56kDa as measured following electrophoresis on an SDS-polyacrylamide gel,wherein the seventh isolated iron-regulated polypeptide comprises theamino acid sequences of SEQ ID NO:822, SEQ ID NO:823, SEQ ID NO:824, SEQID NO:825, SEQ ID NO:826, SEQ ID NO:827, SEQ ID NO:828, SEQ ID NO:829,and SEQ ID NO:830; an eighth isolated iron-regulated polypeptide from E.coli strain BEcO157(stx-), deposited under ATCC accession numberPTA-124618, having a molecular weight of 38 kDa as measured followingelectrophoresis on an SDS-polyacrylamide gel, wherein the eighthisolated iron-regulated polypeptide comprises the amino acid sequencesof SEQ ID NO:831, SEQ ID NO:832, SEQ ID NO:833, SEQ ID NO:834, SEQ IDNO:835, SEQ ID NO:836, SEQ ID NO:837, SEQ ID NO:838, SEQ ID NO:839, SEQID NO:840, SEQ ID NO:841, SEQ ID NO:843, SEQ ID NO:844, SEQ ID NO:847,SEQ ID NO:848, SEQ ID NO:851, SEQ ID NO:853, and SEQ ID NO:852; a ninthisolated iron-regulated polypeptide from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, having a molecularweight of 37 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the ninth isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:854, SEQ IDNO:855, SEQ ID NO:856, SEQ ID NO:857, SEQ ID NO:858, SEQ ID NO:859, SEQID NO:860, SEQ ID NO:861, SEQ ID NO:863, SEQ ID NO:864, SEQ ID NO:866,SEQ ID NO:868, SEQ ID NO:869, SEQ ID NO:870, and SEQ ID NO:872; or atenth isolated iron-regulated polypeptide from E. coli strainBEcO157(stx-), deposited under ATCC accession number PTA-124618, havinga molecular weight of 29 kDa as measured following electrophoresis on anSDS-polyacrylamide gel, wherein the tenth isolated iron-regulatedpolypeptide comprises the amino acid sequences of SEQ ID NO:873, SEQ IDNO:874, SEQ ID NO:875, SEQ ID NO:877, and SEQ ID NO:878.
 15. A method ofinducing production in an animal of antibody that specifically binds aniron regulated polypeptide isolated from E. coli strain BEcO157(stx-),deposited under ATCC accession number PTA-124618, the method comprisingadministering to the animal an effective amount of the composition ofclaim
 13. 16. The method of claim 15 wherein the animal is avian,bovine, caprine, ovine, porcine, bisontine, cervine, equine, a companionanimal, or human.
 17. The method of claim 16 wherein the method furthercomprises collecting the antibody from the animal.
 18. The method ofclaim 17 wherein the method further comprises isolating the antibody.